17ß-Hydroxysteroid Dehydrogenase Type 13 (HSD17B13) iRNA Compositions And Methods Of Use Thereof

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 12, 2019, is named 121301-08420_SL.txt and is 1,374,047 bytes in size.

BACKGROUND OF THE INVENTION

17β-hydroxysteroid dehydrogenase Type 13 (HSD17B13) is a member of the 17β-Hydroxysteroid dehydrogenase (HSD17B) family of enzymes whose members have various functions, including, for example, reduction or oxidation of sex hormones, fatty acids, and bile acids in vivo (Moeller and Adamski (2009) Mol Cell Endocrinol 301:7). Members of the HSD17B family differ in tissue distribution, subcellular localization, catalytic preference, and have diverse substrate specificities as they also catalyze the conversions of other substrates than steroids, as for example lipids and retinoids (Marchais-Oberwinkler, et al. (2011) J Steroid Biochem Mol Biol 125(1-2):66-82). HSD17B13 has been demonstrated to enhance hepatic lipogenesis in normal mouse liver and cultured human hepatocytes (Su, et al. (2014) Proc Natl Acad Sci USA 111:11437).

Hepatocytes, which form the parenchymal tissue of the liver, are responsible for mobilizing lipids for energy and storing excess lipids in the form of lipid droplets (LDs) making the liver the primary organ responsible for lipid homeostasis.

LDs are now recognized as bioactive organelles involved in lipid metabolism, membrane traffic and signal transduction. LDs are generally composed of a core of neutral lipids (such as triacylglcerols (TGs) and cholesterol esters surrounded by a phospholipid/cholesterol monolayer. Numerous LD-specific proteins associate with the membrane of LDs and function, e.g., to control the flux of molecules into and out of the LDs. The predominant hepatocellular LD-associated proteins are members of the perilipin family of proteins, but non-perilipin proteins, such as hypoxia-inducible protein 2 (HIG2), patanin-like phospholipase domain-containing 3 (PNPLA3), and HSD17B13, have also been identified as LD-associate proteins (Carr and Ahima (2016) Exp Cell Res 15:187; Su, et al. (2014) Proc Natl Acad Sci USA 111:11437).

Increased accumulation of LDs is associated with many metabolic diseases and chronic fibro-inflammatory liver diseases, such as liver fibrosis, NASH and NAFLD. HSD17B13 has been identified as one of the most abundantly expressed LD proteins specifically localized on the surface of LDs in human subjects and mice with NAFLD. The level of expression of HSD17B13 was also shown to be up-regulated in the livers of patients and mice with NAFLD. Overexpression of HSD17B13 resulted in an increase in the number and size of LDs. Hepatic overexpression of HSD17B13 in C57BL/6 mice significantly increased lipogenesis and TG contents in the livers, leading to a fatty liver phenotype.

There is currently no treatment for chronic fibro-inflammatory liver diseases. The current standard of care for subjects having a chronic fibro-inflammatory liver disease includes, lifestyle modification and managing the associated comorbidities, e.g., hypertension, hyperlipidemia, diabetes, obesity, etc. Accordingly, as the prevalence of chronic fibro-inflammatory liver diseases has progressively increased over the past 10 years and is expected to increase, there is a need in the art for alternative treatments for subjects having a chronic fibro-inflammatory liver disease.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a 17β-hydroxysteroid dehydrogenase Type 13 (HSD17B13) gene. The HSD17B13 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of a HSD17B13 gene and/or for treating a subject who would benefit from inhibiting or reducing the expression of a HSD17B13 gene, e.g., a subject suffering or prone to suffering from an HSD17B13-associated disease, for example, a chronic fibro-inflammatory liver disease.

Accordingly, in one aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of 17β-hydroxysteroid dehydrogenases type 13 (HSD17B13) in a cell. The dsRNA agent includes a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 or 2 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:8 or 9. In some embodiments, the dsRNA agent includes a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of SEQ ID NO:1 or 2 and the antisense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of SEQ ID NO:8 or 9.

In another aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of 17β-hydroxysteroid dehydrogenases type 13 (HSD17B13) in a cell. The dsRNA agent includes a sense strand and an antisense strand forming a double stranded region, wherein said antisense strand comprises a region of complementarity to an mRNA encoding HSD17B13 which comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from any one of the antisense sequences listed in any one of Tables 2, 3, 7, 8, 10, 11, or 13. In some embodiments, the dsRNA agent includes a sense strand and an antisense strand forming a double stranded region, wherein said antisense strand comprises a region of complementarity to an mRNA encoding HSD17B13 which comprises at least 15 contiguous nucleotides from any one of the antisense sequences listed in any one of Tables 2, 3, 7, 8, 10, 11, or 13.

In one embodiment, the region of complementarity comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from any one of nucleotides 213-242; 256-287; 361-385; 447-480; 483-529; 489-529; 630-653; 688-711; 752-777; 753-779; 772-806; 781-806; 791-851; 829-858; 870-896; 893-930; 900-930; 910-932; 980-1092; 1101-1158; 1176-1210; 1320-1350; 1335-1373; 1456-1482; 1506-1535; 1558-1588; 1699-1740; 1725-1757; 2182-2210; 2190-2254; 2194-2216; 2240-2373; or 2242-2264 of SEQ ID NO:1. In some embodiments, the region of complementarity comprises at least 15 contiguous nucleotides from any one of nucleotides 213-242; 256-287; 361-385; 447-480; 483-529; 489-529; 630-653; 688-711; 752-777; 753-779; 772-806; 781-806; 791-851; 829-858; 870-896; 893-930; 900-930; 910-932; 980-1092; 1101-1158; 1176-1210; 1320-1350; 1335-1373; 1456-1482; 1506-1535; 1558-1588; 1699-1740; 1725-1757; 2182-2210; 2190-2254; 2194-2216; 2240-2373; or 2242-2264 of SEQ ID NO:1.

In another embodiment, the region of complementarity comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from any one of nucleotides 71-93, 101-123, 108-130, 109-131, 112-134, 123-145; 213-235; 220-242, 256-278; 292-314, 293-315, 299-321, 301-323, 324-346, 349-371, 350-372, 351-373, 352-374; 353-375, 355-377, 356-378, 357-379, 361-383; 363-385, 365-387, 402-424, 422-444, 423-445, 427-449, 428-450, 431-453, 447-469, 489-511, 490-512, 507-529, 541-563, 547-569, 548-570, 585-607, 589-611, 592-614, 593-615, 620-642, 630-652, 631-653, 632-654, 649-671, 676-698, 688-710, 723-745, 728-750, 752-774, 753-775, 755-777, 757-779, 763-785, 764-786, 772-794, 778-800, 780-802, 781-803, 791-813, 792-814, 794-816, 795-817, 807-829, 828-850, 829-851; 832-854; 836-858; 838-860; 839-861, 840-862, 832-861; 870-892; 874-896, 894-916; 895-917; 896-918; 897-919; 898-920; 899-921; 900-922; 901-923; 902-924; 906-928; 908-930; 894-930; 910-932; 965-987; 966-988981-1003; 1005-1027; 1006-1028; 1010-1032; 1005-1032; 1052-1074; 1097-1119; 1101-1123; 1102-1124; 1103-1125; 1133-1155; 1135-1157; 1136-1158; 1097-1125; 1133-1158; 1176-1198; 1188-1210; 1243-1265; 1315-13371320-1342; 1322-1344; 1325-1347; 1327-1349; 1328-1350; 1320-1507; 1335-1357; 1336-1358; 1458-1480; 1459-1481; 1460-1482; 1458-1482; 1497-1519; 1498-1520; 1506-1528; 1513-1535; 1565-1587; 1566-1588; 1613-1635; 1614-1636; 1622-1644; 1643-1665; 1699-1721; 1717-1739; 1718-1740; 1724-1746; 1725-1747; 1726-1748; 1727-1749; 1728-1750; 1717-1750; 1737-1759; 1768-1790; 2188-2210; 2190-2212; 2188-2212; 2194-2216; 2195-2217; 2250-2272; 2232-2254; 2240-2262; 2232-2262; 2242-2264; 2245-22672249-2271; 2232-2271; 2347-2369; 2351-2373; or 2347-2373 of SEQ ID NO:1. In some embodiments, the region of complementarity comprises at least 15 contiguous nucleotides from any one of nucleotides 71-93, 101-123, 108-130, 109-131, 112-134, 123-145; 213-235; 220-242, 256-278; 292-314, 293-315, 299-321, 301-323, 324-346, 349-371, 350-372, 351-373, 352-374; 353-375, 355-377, 356-378, 357-379, 361-383; 363-385, 365-387, 402-424, 422-444, 423-445, 427-449, 428-450, 431-453, 447-469, 489-511, 490-512, 507-529, 541-563, 547-569, 548-570, 585-607, 589-611, 592-614, 593-615, 620-642, 630-652, 631-653, 632-654, 649-671, 676-698, 688-710, 723-745, 728-750, 752-774, 753-775, 755-777, 757-779, 763-785, 764-786, 772-794, 778-800, 780-802, 781-803, 791-813, 792-814, 794-816, 795-817, 807-829, 828-850, 829-851; 832-854; 836-858; 838-860; 839-861, 840-862, 832-861; 870-892; 874-896, 894-916; 895-917; 896-918; 897-919; 898-920; 899-921; 900-922; 901-923; 902-924; 906-928; 908-930; 894-930; 910-932; 965-987; 966-988981-1003; 1005-1027; 1006-1028; 1010-1032; 1005-1032; 1052-1074; 1097-1119; 1101-1123; 1102-1124; 1103-1125; 1133-1155; 1135-1157; 1136-1158; 1097-1125; 1133-1158; 1176-1198; 1188-1210; 1243-1265; 1315-13371320-1342; 1322-1344; 1325-1347; 1327-1349; 1328-1350; 1320-1507; 1335-1357; 1336-1358; 1458-1480; 1459-1481; 1460-1482; 1458-1482; 1497-1519; 1498-1520; 1506-1528; 1513-1535; 1565-1587; 1566-1588; 1613-1635; 1614-1636; 1622-1644; 1643-1665; 1699-1721; 1717-1739; 1718-1740; 1724-1746; 1725-1747; 1726-1748; 1727-1749; 1728-1750; 1717-1750; 1737-1759; 1768-1790; 2188-2210; 2190-2212; 2188-2212; 2194-2216; 2195-2217; 2250-2272; 2232-2254; 2240-2262; 2232-2262; 2242-2264; 2245-22672249-2271; 2232-2271; 2347-2369; 2351-2373; or 2347-2373 of SEQ ID NO:1.

In another embodiment, the region of complementarity comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from any one of nucleotides 108-130; 109-131; 108-131; 112-134; 293-315; 301-323; 293-323; 361-383; 402-424; 423-445; 428-450; 423-450; 428-453; 431-453; 489-511; 490-512; 489-512; 649-671; 753-775; 772-794; 791-813; 792-814; 795-817; 791-817; 829-851; 832-854; 836-858; 829-858; 870-892; 874-896; 870-896; 898-920; 900-922; 902-924; 906-928; 908-930; 902-930; 910-932; 966-988; 1328-1350; or 2194-2216; 2242-2264; or 2249-2271 of SEQ ID NO:1. In some embodiments, the region of complementarity comprises at least 15 contiguous nucleotides from any one of nucleotides 108-130; 109-131; 108-131; 112-134; 293-315; 301-323; 293-323; 361-383; 402-424; 423-445; 428-450; 423-450; 428-453; 431-453; 489-511; 490-512; 489-512; 649-671; 753-775; 772-794; 791-813; 792-814; 795-817; 791-817; 829-851; 832-854; 836-858; 829-858; 870-892; 874-896; 870-896; 898-920; 900-922; 902-924; 906-928; 908-930; 902-930; 910-932; 966-988; 1328-1350; or 2194-2216; 2242-2264; or 2249-2271 of SEQ ID NO:1. In another embodiment, the region of complementarity comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from nucleotides 898-930 of SEQ ID NO:1. In some embodiments, the region of complementarity comprises at least 15 contiguous nucleotides from nucleotides 898-930 of SEQ ID NO:1.

In one embodiment, the region of complementarity comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from nucleotides 910-932 of SEQ ID NO:1. In some embodiments, the region of complementarity comprises at least 15 contiguous nucleotides from nucleotides 910-932 of SEQ ID NO:1.

In one embodiment, the region of complementarity comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from nucleotides 2194-2216 of SEQ ID NO:1. In some embodiments, the region of complementarity comprises at least 15 contiguous nucleotides from nucleotides 2194-2216 of SEQ ID NO:1.

In one embodiment, the region of complementarity comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from nucleotides 2242-2264 of SEQ ID NO:1. In some embodiments, the region of complementarity comprises at least 15 contiguous nucleotides from nucleotides 2242-2264 of SEQ ID NO:1.

In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

In one embodiment, substantially all of the nucleotides of the sense strand comprise a modification. In another embodiment, substantially all of the nucleotides of the antisense strand comprise a modification. In yet another embodiment, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand comprise a modification.

In one aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of 17β-hydroxysteroid dehydrogenases type 13 (HSD17B13) in a cell. The dsRNA agent includes a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 or 2 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:8 or 9, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3′-terminus. In some embodiments, the dsRNA agent includes a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of SEQ ID NO:1 or 2 and the antisense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of SEQ ID NO:8 or 9, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3′-terminus.

In one embodiment, all of the nucleotides of the sense strand comprise a modification. In another embodiment, all of the nucleotides of the antisense strand comprise a modification. In yet another embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In one embodiment, at least one of said modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a glycol modified nucleotide, and a 2-O—(N-methylacetamide) modified nucleotide, and combinations thereof.

In one embodiment, the nucleotide modifications are 2′-O-methyl and/or 2′-fluoro modifications.

The region of complementarity may be at least 17 nucleotides in length; 19 to 30 nucleotides in length; 19-25 nucleotides in length; or 21 to 23 nucleotides in length.

Each strand may be no more than 30 nucleotides in length, e.g., each strand is independently 19-30 nucleotides in length; each strand is independently 19-25 nucleotides in length; each strand is independently 21-23 nucleotides in length.

The dsRNA may include at least one strand that comprises a 3′ overhang of at least 1 nucleotide; or at least one strand that comprises a 3′ overhang of at least 2 nucleotides.

In some embodiment, the dsRNA agent further comprises a ligand.

In one embodiment, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA agent.

In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

In one embodiment, the ligand is

embedded image

In one embodiment, the dsRNA agent is conjugated to the ligand as shown in the following schematic

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and, wherein X is O or S.

In one embodiment, the X is O.

In one embodiment, the region of complementarity comprises any one of the antisense sequences in any one of Tables 2, 3, 7, 8, 10, 11, or 13.

In one aspect, the present invention provides a double stranded for inhibiting expression of 17β-hydroxysteroid dehydrogenases type 13 (HSD17B13) in a cell. The dsRNA agent includes a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding HSD17B13, wherein each strand is about 14 to about 30 nucleotides in length, wherein said dsRNA agent is represented by formula (III):

sense: 5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′

antisense: 3′n_p′-N_a′—(X′X′X′)_k—N_b′—Y′Y′Y′—N_b′—(Z′Z′Z′)_l—N_a′-n_q′5′ (III)

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides;

- each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof;
- each n_p, n_p′, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide;
  - XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides;
  - modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and
  - wherein the sense strand is conjugated to at least one ligand.

In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1. In another embodiment, k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1.

In one embodiment, XXX is complementary to X′X′X′, YYY is complementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.

In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand, e.g., the Y′Y′Y′ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′-end.

In one embodiment, formula (III) is represented by formula (IIIa):

sense: 5′n_p-N_a—YYY—N_a-n_q3′

antisense: 3′n_p′-N_a′—Y′Y′Y′—N_a′-n_q′5′ (IIIa).

In another embodiment, formula (III) is represented by formula (IIIb):

sense: 5′n_p-N_a—YYY—N_b—ZZZ—N_a-n_q3′

antisense: 3′n_p′-N_a′—Y′Y′Y′—N_b′—Z′Z′Z′—N_a′-n_q′5′ (IIIb)

wherein each N_band N_b′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

In yet another embodiment, formula (III) is represented by formula (IIIc):

sense: 5′n_p-N_a—XXX—N_b—YYY—N_a-n_q3′

antisense: 3′n_p′-N_a′—X′X′X′—N_b′—Y′Y′Y′—N_a′-n_q′5′ (IIIc)

wherein each N_band N_b′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

In another embodiment, formula (III) is represented by formula (IIId):

sense: 5′n_p-N_a—XXX—N_b—YYY—N_b—ZZZ—N_a-n_q3′

antisense: 3′n_p′-N_a′—X′X′X′—N_b′—Y′Y′Y′—N_b′—Z′Z′Z′—N_a′-n_q′5′ (IIId)

wherein each N_band N_b′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides and each N_aand N_a′ independently represents an oligonucleotide sequence comprising 2-10 modified nucleotides.

The region of complementarity may be at least 17 nucleotides in length; 19 to 30 nucleotides in length; 19-25 nucleotides in length; or 21 to 23 nucleotides in length.

Each strand may be no more than 30 nucleotides in length, e.g., each strand is independently 19-30 nucleotides in length.

In one embodiment, the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-deoxy, 2′-hydroxyl, and combinations thereof.

In one embodiment, the modifications on the nucleotides are 2′-O-methyl or 2′-fluoro modifications.

In one embodiment, the Y′ is a 2′-O-methyl or 2′-fluoro modified nucleotide.

In one embodiment, at least one strand of the dsRNA agent may comprise a 3′ overhang of at least 1 nucleotide; or a 3′ overhang of at least 2 nucleotides.

In one embodiment, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand. In one embodiment, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand. In one embodiment, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

In one embodiment, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at both the 5′- and 3′-terminus of one strand.

In one embodiment, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

In one embodiment, p′>0. In another embodiment, p′=2.

In one embodiment, q′=0, p=0, q=0, and p′ overhang nucleotides are complementary to the target mRNA. In another embodiment, q′=0, p=0, q=0, and p′ overhang nucleotides are non-complementary to the target mRNA.

In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

In one embodiment, at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage. In another embodiment, wherein all n_p′ are linked to neighboring nucleotides via phosphorothioate linkages.

In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In one embodiment, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA agent.

In one embodiment, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached through a monovalent, bivalent, or trivalent branched linker.

In one embodiment, the ligand is

embedded image

In one embodiment, the dsRNA agent is conjugated to the ligand as shown in the following schematic

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and, wherein X is O or S.

In one embodiment, the X is O.

sense: 5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′

antisense: 3′n_p′-N_a′—(X′X′X′)_k—N_b′—Y′Y′Y′—N_b′—(Z′Z′Z′)_l—N_a′-n_q′5′ (III)

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

- each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof;

each n_p, n_p′, n_q, and n_q′, each of which may or may not be present independently represents an overhang nucleotide;

- XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications;
- modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and
- wherein the sense strand is conjugated to at least one ligand.

sense: 5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′

antisense: 3′n_p′-N_a′—(X′X′X′)_k—N_b′—Y′Y′Y′—N_b′—(Z′Z′Z′)_l—N_a′-n_q′5′ (III)

wherein:

i, j, k, and l are each independently 0 or 1;

each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage;

- each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof;
- XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications;
  - modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and
  - wherein the sense strand is conjugated to at least one ligand.

sense: 5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′

antisense: 3′n_p′-N_a′—(X′X′X′)_k—N_b′—Y′Y′Y′—N_b′—(Z′Z′Z′)_l—N_a′-n_q′5′ (III)

wherein:

i, j, k, and l are each independently 0 or 1;

each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage;

- each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications;

- modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and
- wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, bivalent, or trivalent branched linker.

sense: 5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′

antisense: 3′n_p′-N_a′—(X′X′X′)_k—N_b′—Y′Y′Y′—N_b′—(Z′Z′Z′)_l—N_a′-n_q′5′ (III)

wherein:

i, j, k, and l are each independently 0 or 1;

- each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage;

- each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof;

- modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′;
- wherein the sense strand comprises at least one phosphorothioate linkage; and

wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, bivalent, or trivalent branched linker.

sense: 5′n_p-N_a—YYY—N_a-n_q3′

antisense: 3′n_p′-N_a′—Y′Y′Y′—N_a′-n_q′5′ (IIIa)

wherein:

- each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide;
- p, q, and q′ are each independently 0-6;
- n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage;
- each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides;
- YYY and Y′Y′Y′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl and/or 2′-fluoro modifications;
- wherein the sense strand comprises at least one phosphorothioate linkage; and
- wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, bivalent, or trivalent branched linker.

In one aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of 17β-hydroxysteroid dehydrogenases type 13 (HSD17B13) in a cell. The dsRNA agent includes a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 or 2 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:8 or 9, wherein substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus, wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus, and wherein the sense strand is conjugated to one or more GalNAc derivatives attached through a monovalent, bivalent or trivalent branched linker at the 3′-terminus. In some embodiments, the dsRNA agent includes a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of SEQ ID NO:1 or 2 and the antisense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of SEQ ID NO:8 or 9, wherein substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus, wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus, and wherein the sense strand is conjugated to one or more GalNAc derivatives attached through a monovalent, bivalent or trivalent branched linker at the 3′-terminus.

In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

In one embodiment, the region of complementarity comprises any one of the antisense sequences listed in any one of Tables 2, 3, 7, 8, 10, 11, or 13. In one embodiment, the agent is selected from the group consisting of AD-288917, AD-288996, AD-413639, AD-413644, and AD-413669. In one embodiment, the RNAi agent is AD-288917. In another embodiment, the agent is AD-288996. In another embodiment, the agent is AD-413639. In one embodiment, the agent is AD-413644. In another embodiment, the agent is AD-413669

In one embodiment, the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of the nucleotide sequences of any one of the agents listed in any one of Tables 2, 3, 7, 8, 10, 11, or 13.

The present invention also provides cells, vectors, and pharmaceutical compositions which include any of the dsRNA agents of the invention. The dsRNA agents mat be forumated in an unbuffered solution, e.g., saline or water, or in a buffered solution, e.g., a solution comprising acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In one embodiment, the buffered solution is phosphate buffered saline (PBS).

In one aspect, the present invention provides a method of inhibiting 17β-hydroxysteroid dehydrogenases type 13 (HSD17B13) expression in a cell. The method includes contacting the cell with a dsRNA agent or a pharmaceutical composition of the invention, thereby inhibiting expression of HSD17B13 in the cell.

The cell may be within a subject, such as a human subject.

In one embodiment, the HSD17B13 expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or to below the level of detection of HSD17B13 expression.

In one embodiment, the human subject suffers from an HSD17B13-associated disease, disorder, or condition. In one embodiment, the HSD17B13-associated disease, disorder, or condition is a chronic fibro-inflammatory liver disease. In one embodiment, the chronic fibro-inflammatory liver disease is selected from the group consisting of inflammation of the liver, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), cirrhosis of the liver, alcoholic steatohepatitis (ASH), alcoholic liver diseases (ALD), HCV-associated cirrhosis, drug induced liver injury, and hepatocellular necrosis.

In one aspect, the present invention provides a method of inhibiting the expression of HSD17B13 in a subject. The methods include administering to the subject a therapeutically effective amount of a dsRNA agent or a pharmaceutical composition of the invention, thereby inhibiting the expression of HSD17B13 in the subject.

In another aspect, the present invention provides a method of treating a subject suffering from an HSD17B13-associated disease, disorder, or condition. The method includes administering to the subject a therapeutically effective amount of of a dsRNA agent or a pharmaceutical composition of the invention, thereby treating the subject suffering from an HSD17B13-associated disease, disorder, or condition.

In another aspect, the present invention provides a method of preventing at least one symptom in a subject having a disease, disorder or condition that would benefit from reduction in expression of an HSD17B13 gene. The method includes administering to the subject a prophylactically effective amount of the agent of a dsRNA agent or a pharmaceutical composition of the invention, thereby preventing at least one symptom in a subject having a disease, disorder or condition that would benefit from reduction in expression of an HSD17B13 gene.

In another aspect, the present invention provides a method of reducing the risk of developing chronic liver disease in a subject having steatosis. The method includes administering to the subject a therapeutically effective amount of a dsRNA agent or a pharmaceutical composition of the invention, thereby reducing the risk of developing chronic liver disease in the subject having steatosis.

In yet another aspect, the present invention provides a method of inhibiting the progression of steatosis to steatohepatitis in a subject suffering from steatosis. The method includes administering to the subject a therapeutically effective amount of a dsRNA agent or a pharmaceutical composition of the invention, thereby inhibiting the progression of steatosis to steatohepatitis in the subject.

In one aspect, the present invention provides a method of inhibiting the accumulation of lipid droplets in the liver of a subject suffering from an HSD17B13-associated disease, disorder, or condition. The method includes administering to the subject a therapeutically effective amount of a dsRNA agent or a pharmaceutical composition of the invention, and a dsRNA agent targeting a PNPLA3 gene or a pharmaceutical composition comprising a dsRNA agent targeting a PNPLA3 gene, thereby inhibiting the accumulation of fat in the liver of the subject suffering from an HSD17B13-associated disease, disorder, or condition.

In another aspect, the present invention provides a method of treating a subject suffering from an HSD17B13-associated disease, disorder, or condition. The method includes administering to the subject a therapeutically effective amount of a dsRNA agent or a pharmaceutical composition of the invention, and a dsRNA agent targeting a PNPLA3 gene or a pharmaceutical composition comprising a dsRNA agent targeting a PNPLA3 gene, thereby treating the subject suffering from an HSD17B13-associated disease, disorder, or condition.

In another aspect, the present invention provides a method of preventing at least one symptom in a subject having a disease, disorder or condition that would benefit from reduction in expression of an HSD17B13 gene. The method includes administering to the subject a therapeutically effective amount of a dsRNA agent or a pharmaceutical composition of the invention, and a dsRNA agent targeting a PNPLA3 gene or a pharmaceutical composition comprising a dsRNA agent targeting a PNPLA3 gene, thereby preventing at least one symptom in a subject having a disease, disorder or condition that would benefit from reduction in expression of an HSD17B13 gene.

In another aspect, the present invention provides a method of reducing the risk of developing chronic liver disease in a subject having steatosis. The method includes administering to the subject a therapeutically effective amount of a dsRNA agent or a pharmaceutical composition of the invention, and a dsRNA agent targeting a PNPLA3 gene or a pharmaceutical composition comprising a dsRNA agent targeting a PNPLA3 gene, thereby reducing the risk of developing chronic liver disease in the subject having steatosis.

In another aspect, the present invention provides a method of inhibiting the progression of steatosis to steatohepatitis in a subject suffering from steatosis. The method includes administering to the subject a therapeutically effective amount of a dsRNA agent or a pharmaceutical composition of the invention, and a dsRNA agent targeting a PNPLA3 gene or a pharmaceutical composition comprising a dsRNA agent targeting a PNPLA3 gene, thereby inhibiting the progression of steatosis to steatohepatitis in the subject.

In one embodiment, the administration of the dsRNA agent or the pharmaceutical composition to the subject causes a decrease in HSD17B13 enzymatic activity, a decrease in HSD17B13 protein accumulation, a decrease in PNPLA3 enzymatic activity, a decrease in PNPLA3 protein accumulation, and/or a decrease in accumulation of fat and/or expansion of lipid droplets in the liver of a subject.

In one embodiment, the HSD17B13-associated disease, disorder, or condition is a chronic fibro-inflammatory liver disease.

In one embodiment, the chronic fibro-inflammatory liver disease is selected from the group consisting of accumulation of fat in the liver, inflammation of the liver, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), cirrhosis of the liver, alcoholic steatohepatitis (ASH), alcoholic liver diseases (ALD), HCV-associated cirrhosis, drug induced liver injury, and hepatocellular necrosis.

In one embodiment, the chronic fibro-inflammatory liver disease is nonalcoholic steatohepatitis (NASH).

In one embodiment, the subject is obese.

In one embodiment, the methods and uses of the invention further include administering an additional therapeutic to the subject.

In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.

The agent may be administered to the subject intravenously, intramuscularly, or subcutaneously. In one embodiment, the agent is administered to the subject subcutaneously.

In one embodiment, the methods and uses of the invention further include determining, the level of HSD17B13 in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that HSD17B13 rs72613567:TA is associated with reduced risk of alcoholic and nonalcoholic liver disease phenotypes. Specifically, FIG. 1 shows HSD17B13 rs72613567:TA was associated with lower odds of various chronic liver diseases in an allele dosage-dependent manner. Specifically, allele dosage-dependent effects were observed for both alcoholic and non-alcoholic liver diseases, cirrhosis and hepatocellular carcinoma. Odds ratios were calculated using logistic regression, with adjustment for age, gender, BMI, and self-reported ethnicity.

FIG. 2A is a graph depicting the effect of a single dose of AD-288917 in mice expressing human HSD17B13.

FIG. 2B is a graph depicting the effect of a single dose of AD-288917 in Cynomolgus monkeys.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions, which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an HSD17B13 gene. The HSD17B13 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of an HSD17B13 gene, and for treating a subject who would benefit from inhibiting or reducing the expression of an HSD17B13 gene, e.g., a subject that would benefit from a reduction in inflammation of the liver, e.g., a subject suffering or prone to suffering from an HSD17B13-associated disease disorder, or condition, such as a subject suffering or prone to suffering from liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), alcoholic liver diseases (ALD), cirrhosis of the liver, HCV-associated cirrhosis, drug induced liver injury, and hepatocellular necrosis.

The iRNAs of the invention targeting HSD17B13 may include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an HSD17B13 gene.

In some embodiments, one or both of the strands of the double stranded RNAi agents of the invention is up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA transcript of an HSD17B13 gene. In some embodiments, such iRNA agents having longer length antisense strands may include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

The use of the iRNA agents described herein enables the targeted degradation of mRNAs of an HSD17B13 gene in mammals.

Very low dosages of the iRNAs, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of an HSD17B13 gene. Thus, methods and compositions including these iRNAs are useful for treating a subject who would benefit from inhibiting or reducing the expression of an HSD17B13 gene, e.g., a subject that would benefit from a reduction of inflammation of the liver, e.g., a subject suffering or prone to suffering from an HSD17B13-associated disease disorder, or condition, such as a subject suffering or prone to suffering from liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), alcoholic liver diseases (ALD), cirrhosis of the liver, HCV-associated cirrhosis, drug induced liver injury, and hepatocellular necrosis.

The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of an HSD17B13 gene, as well as compositions and methods for treating subjects having diseases and disorders that would benefit from inhibition and/or reduction of the expression of this gene.

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means±10%. In certain embodiments, about means±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

The term “HSD17B13,” also known as “hydroxysteroid 17-beta dehydrogenase 13,” “short chain dehydrogenase/reductase family 16C member,” “short-chain dehydrogenase/reductase 9,” “17-beta-HSD 13,” “17P-HSD13,” “SDR16C3,” “SCDR9,” “short chain dehydrogenase/reductase family 16C, Member 3,” “hydroxysteroid (17-beta) dehydrogenase 13,” “17-beta-hydroxysteroid dehydrogenase 13,” “17-beta hydroxysteroid dehydrogenase,” “HMFN0376,” and “NIIL497,” refers to the well known gene encoding a 17β-hydroxysteroid dehydrogenase type 13 protein from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless specified otherwise.

The term also refers to fragments and variants of native HSD17B13 that maintain at least one in vivo or in vitro activity of a native HSD17B13. The term encompasses full-length unprocessed precursor forms of HSD17B13 as well as mature forms resulting from post-translational cleavage of the signal peptide and forms resulting from proteolytic processing.

Two variants of the human HSD17B13 gene were previously identified, variant A (or Transcript A) and variant B (or Transcript B). Transcript A includes all seven exons of the HSD17B13 gene, whereas exon 2 is skipped in Transcript B. The nucleotide and amino acid sequence of a human HSD17B13 variant A can be found in, for example, GenBank Reference Sequence: NM_178135.4; SEQ ID NO:1); and the nucleotide and amino acid sequence of a human HSD17B13 variant B can be found in, for example, GenBank Reference Sequence: NM_001136230.2; SEQ ID NO:2. As described in U.S. patent application Ser. No. 15/875,514, filed on Jan. 19, 2018, and PCT Application No.: PCT/US2018/014357, filed on Jan. 19, 2018 (the entire contents of each of which are incorporated herein by reference), six additional HSD17B13 transcripts that are expressed (C—H, SEQ ID NOS: 17, 18, 19, 20, 21, and 22, respectively) have been identified. In Transcript C, exon 6 is skipped compared to Transcript A. In Transcript D, there is an insertion of a guanine 3′ of exon 6, resulting in a frameshift in and premature truncation of exon 7 compared to Transcript A. In Transcript E, there is an additional exon between exons 3 and 4 compared to Transcript A. In Transcript F, which is expressed only in HSD17B13 rs72613567 variant carriers, there is read-through from exon 6 into intron 6 compared to Transcript A. In Transcript G, exon 2 is skipped, and there is an insertion of a guanine 3′ of exon 6, resulting in a frameshift in and premature truncation of exon 7 compared to Transcript A. In Transcript H, there is an additional exon between exons 3 and 4, and there is an insertion of a guanine 3′ of exon 6, resulting in a frameshift in and premature truncation of exon 7 compared to Transcript A.

One additional HSD17B13 transcript that is expressed at low levels (F′, SEQ ID NO: 23) has also been identified. Like Transcript F, Transcript F′ also includes a read-through from exon 6 into intron 6 compared to Transcript A, but, in contrast to Transcript F, the read-through does not include the inserted thymine present in the HSD17B13 rs72613567 variant gene. The nucleotide positions of the exons within the HSD17B13 genes for each Transcript are provided below.

SEQ ID NO:15 is the nucleotide sequence of the HSD17B13 Wild Type Genomic Sequence (Human Genome Assembly GRCh38) and SEQ ID NO: 16 is the nucleotide sequence of HSD17B13 Genomic Sequence Variant (Human Genome Assembly GRCh38; rs72613567—insertion of T at chr4: 87310241-87310240): Insertion of T at position 12666.

Nucleotide Positions in SEQ ID NO: 15 for Exons

of HSD17B13 Transcripts More Prevalent in Subjects

Homozygous for Wild Type HSD17B13 Gene.

Transcript A
Transcript B
Transcript E
Transcript F′

Exon 1
1-275
1-275
1-275
1-275

Exon 2
4471-4578
skipped
4471-4578
4471-4578

Exon 3
5684-5815
5684-5815
5684-5815
5684-5815

Exon 3′
not present
not present
6210-6281
not present

Exon 4
7308-7414
7308-7414
7308-7414
7308-7414

Exon 5
8947-9084
8947-9084
8947-9084
8947-9084

Exon 6
12548-12664
12548-12664
12548-12664
12548-13501*

Exon 7
17599-19118
17599-19118
17599-19118
skipped

*includes read-through from exon 6 into intron 6; read-through = positions 12665-13501

Nucleotide Positions in SEQ ID NO: 16 for Exons of HSD17B13 Transcripts

More Prevalent in Subjects Homozygous for rs72613567 HSD17B13

Variant Gene (Insertion of T at Position 12666).

Transcript C
Transcript D
Transcript F
Transcript G
Transcript H

Exon 1
1-275
1-275
1-275
1-275
1-275

Exon 2
4471-4578
4471-4578
4471-4578
skipped
4471-4578

Exon 3
5684-5815
5684-5815
5684-5815
5684-5815
5684-5815

Exon 3′
not present
not present
not present
not present
6210-6281

Exon 4
7308-7414
7308-7414
7308-7414
7308-7414
7308-7414

Exon 5
8947-9084
8947-9084
8947-9084
8947-9084
8947-9084

Exon 6
skipped
12548-12665{circumflex over ( )}
12548-13502*
12548-12665{circumflex over ( )}
12548-12665{circumflex over ( )}

Exon 7
17600-19119
17600-19119
skipped
17600-19119
17600-19119

{circumflex over ( )}Includes additional residue 12665 at 3′ end compared to Transcript A

*Includes read-through from exon 6 into intron 6; read-through = positions 12665-13502

There are two variants of the mouse HSD17B13 gene; the nucleotide and amino acid sequence of a mouse Hsd17b13, transcript variant 1 can be found in, for example, GenBank Reference Sequence: NM_001163486.1; SEQ ID NO:3); and the nucleotide and amino acid sequence of a mouse Hsd17b13, transcript variant 2 can be found in, for example, GenBank Reference Sequence: NM_198030.2; SEQ ID NO:4. The nucleotide and amino acid sequence of a rat Hsd17b13 gene can be found in, for example, GenBank Reference Sequence: NM_001009684.1; SEQ ID NO:5). The nucleotide and amino acid sequence of a Macaca mulatta HSD17B13 gene can be found in, for example, GenBank Reference Sequence: XM_015138766.1; SEQ ID NO:6). The nucleotide and amino acid sequence of a Macaca fascicularis HSD17B13 gene can be found in, for example, GenBank Reference Sequence: XM_005555367.2; SEQ ID NO:7).

Additional examples of HSD17B13 mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM.

The term “HSD17B13” as used herein also refers to a particular polypeptide expressed in a cell by naturally occurring DNA sequence variations of the HSD17B13 gene, such as a single nucleotide polymorphism in the HSD17B13 gene. Numerous SNPs within the HSD17B13 gene have been identified and may be found at, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/snp).

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a HSD17B13 gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a HSD17B13 gene.

The target sequence of an HSD17B13 gene may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 1). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of HSD17B13 gene in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a single stranded RNA that interacts with a target RNA sequence, e.g., an HSD17B13 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (sssiRNA) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., an HSD17B13 gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded RNAi agent that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents (ssRNAi) bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAi agents are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, an “iRNA” for use in the compositions and methods of the invention is a double-stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., an HSD17B13 gene. In some embodiments of the invention, a double-stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides. As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, and/or a modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims.

The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.

In one embodiment, an RNAi agent of the invention is a dsRNA, each strand of which comprises less than 30 nucleotides, e.g., 17-27, 19-27, 17-25, 19-25, or 19-23, that interacts with a target RNA sequence, e.g., an HSD17B13 target mRNA sequence, to direct the cleavage of the target RNA. In another embodiment, an RNAi agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence, e.g., an HSD17B13 target mRNA sequence, to direct the cleavage of the target RNA. In one embodiment, the sense strand is 21 nucleotides in length. In another embodiment, the antiosense strand is 23 nucleotides in length.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the extended overhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., an HSD17B13 mRNA.

As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., an HSD17B13 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding HSD17B13). For example, a polynucleotide is complementary to at least a part of an HSD17B13 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding HSD17B13.

Accordingly, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target HSD17B13 sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target HSD17B13 sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:1, or a fragment of SEQ ID NO:1, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target HSD17B13 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:8, or a fragment of any one of SEQ ID NO:8, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In some embodiments, an iRNA of the invention includes an antisense strand that is substantially complementary to the target HSD17B13 sequence and comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of the sense strands in any one of Tables 2, 3, 7, 8, 10, 11, or 13, or a fragment of any one of the sense strands in any one of Tables 2, 3, 7, 8, 10, 11, or 13, such as about about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition.

The phrase “inhibiting expression of an HSD17B13 gene,” as used herein, includes inhibition of expression of any HSD17B13 gene (such as, e.g., a mouse HSD17B13 gene, a rat HSD17B13 gene, a monkey HSD17B13 gene, or a human HSD17B13 gene) as well as variants or mutants of an HSD17B13 gene that encode an HSD17B13 protein.

“Inhibiting expression of an HSD17B13 gene” includes any level of inhibition of an HSD17B13 gene, e.g., at least partial suppression of the expression of an HSD17B13 gene, such as an inhibition by at least about 20%. In certain embodiments, inhibition is by at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

The expression of an HSD17B13 gene may be assessed based on the level of any variable associated with HSD17B13 gene expression, e.g., HSD17B13 mRNA level or HSD17B13 protein level. The expression of an HSD17B13 gene may also be assessed indirectly based on, for example, the levels of circulating alanine aminotransferase (ALT), or the enzymatic activity of HSD17B13 in a tissue sample, such as a liver sample. Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In one embodiment, at least partial suppression of the expression of an HSD17B13 gene, is assessed by a reduction of the amount of HSD17B13 mRNA which can be isolated from, or detected, in a first cell or group of cells in which an HSD17B13 gene is transcribed and which has or have been treated such that the expression of an HSD17B13 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).

The degree of inhibition may be expressed in terms of:

$\frac{(mRNA in control cells) - (mRNA in treated cells)}{(mRNA in control cells)} \cdot 100 %$

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain and/or be coupled to a ligand, e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.

In one embodiment, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be done by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose).

In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in HSD17B13 expression; a human at risk for a disease, disorder or condition that would benefit from reduction in HSD17B13 expression; a human having a disease, disorder or condition that would benefit from reduction in HSD17B13 expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in HSD17B13 expression as described herein.

In one embodiment, the subject is heterozygous for the gene encoding the patatin like phospholipase domain containg 3 (PNPLA3) I148M variation. In another embodiment, the subject is homozygous for the gene encoding the PNPLA3 I148M variation. In one embodiment, the subject is heterozygous for the gene encoding the patatin like phospholipase domain containg 3 (PNPLA3) I144M variation. In another embodiment, the subject is homozygous for the gene encoding the PNPLA3 I144M variation. In one embodiment, the subject is homozygous for the gene encoding a functional HSD17B13 protein. In another embodiment, the subject is heterozygous for the gene encoding a functional HSD17B13 protein. In yet another embodiment, the subject is heterozygous for the gene encoding a functional HSD17B13 protein and a gene encoding a loss of function variant of HSD17B13. In another embodiment, the subject is not a carrier of the HSD17B13 rs72613567 variant, e.g., HSD17B13 rs72613567:TA.

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms associated with HSD17B13 gene expression and/or HSD17B13 protein production, e.g., an HSD17B13-associated disease, such as a chronic fibro-inflammatory liver disease, e.g., inflammation of the liver, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), cirrhosis of the liver, alcoholic steatohepatitis (ASH), alcoholic liver diseases (ALD), HCV-associated cirrhosis, drug induced liver injury, hepatocellular necrosis, and/or hepatocellular carcinoma. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.

The term “lower” in the context of an HSD17B13-associated disease refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more. In certain embodiments, a decrease is at least 20%. “Lower” in the context of the level of HSD17B13 in a subject is preferably down to a level accepted as within the range of normal for an individual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of an HSD17B13 gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with such disease, disorder, or condition, e.g., a symptom of HSD17B13 gene expression, such as inflammation of the liver, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), cirrhosis of the liver, alcoholic steatohepatitis (ASH), alcoholic liver diseases (ALD), HCV-associated cirrhosis, drug induced liver injury, hepatocellular necrosis, and/or hepatocellular carcinoma. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms (e.g., reduction in lipid accumulation in the liver and/or lipid droplet expansion in the liver) delayed (e.g., by days, weeks, months or years) is considered effective prevention.

As used herein, the term “HSD17B13-associated disease,” is a disease or disorder that is caused by, or associated with, HSD17B13 gene expression or HSD17B13 protein production. The term “HSD17B13-associated disease” includes a disease, disorder or condition that would benefit from a decrease in HSD17B13 gene expression or protein activity.

In one embodiment, an “HSD17B13-associated disease” is a chronic fibro-inflammatory liver disease. A “chronic fibro-inflammatory liver disease” is any disease, disorder, or condition associated with chronic liver inflammation and/or fibrosis. Non-limiting examples of a chronic fibro-inflammatory liver disease include, for example, inflammation of the liver, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), cirrhosis of the liver, alcoholic steatohepatitis (ASH), alcoholic liver diseases (ALD), HCV-associated cirrhosis, drug induced liver injury, hepatocellular necrosis, and/or hepatocellular carcinoma.

“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having an HSD17B13-associated disease, disorder, or condition, is sufficient to effective treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a subject having an HSD17B13-associated disease, disorder, or condition, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the iRNA, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylactically effective amount” also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. iRNA employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In some embodiments, a “sample derived from a subject” refers to blood or plasma drawn from the subject.

II. iRNAs of the Invention

Described herein are iRNAs which inhibit the expression of a target gene. In one embodiment, the iRNAs inhibit the expression of an HSD17B13 gene. In one embodiment, the iRNA agent includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an HSD17B13 gene in a cell, such as a liver cell, such as a liver cell within a subject, e.g., a mammal, such as a human having a chronic fibro-inflammatory liver disease, disorder, or condition, e.g., a disease, disorder, or condition associated with, e.g., accumulation and/or expansion of lipid droplets in the liver and/or fibrosis of the liver.

The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an HSD17B13 gene. The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the target gene, the iRNA inhibits the expression of the target gene (e.g., a human, a primate, a non-primate, or a bird target gene) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of an HSD17B13 gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs in length, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence is between 15 and 30 nucleotides in length, e.g., between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

In some embodiments, the sense and antisense strands of the dsRNA are each independently about 15 to about 30 nucleotides in length, or about 25 to about 30 nucleotides in length, e.g., each strand is independently between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In some embodiments, the dsRNA is between about 15 and about 23 nucleotides in length, or between about 25 and about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target HSD17B13 expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand sequence is selected from the group of sequences provided in any one of Tables 2, 3, 7, 8, 10, 11 or 13, and the corresponding nucleotide sequence of the antisense strand of the sense strand is selected from the group of sequences of any one of Tables 2, 3, 7, 8, 10, 11, or 13. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an HSD17B13 gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand (passenger strand) in any one of Tables 2, 3, 7, 8, 10, 11, or 13, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) of the sense strand in any one of Tables 2, 3, 7, 8, 10, 11, or 13. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

It will be understood that, although the sequences in Tables 2, 3, 7, 8, 10, 11, or 13 are described as modified, unmodified, unconjugated. and/or conjugated sequences, the RNA of the iRNA of the invention e.g., a dsRNA of the invention, may comprise any one of the sequences set forth in any one of Tables 2, 3, 7, 8, 10, 11, or 13 that is un-modified, un-conjugated, and/or modified and/or conjugated differently than described therein.

The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided herein, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein, and differing in their ability to inhibit the expression of an HSD17B13 gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.

In addition, the RNAs described in any one of Tables 2, 3, 7, 8, 10, 11, or 13 identify a site(s) in an HSD17B13 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within this site(s). As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in the gene.

While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified herein represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified herein, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of an HSD17B13 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an HSD17B13 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of an HSD17B13 gene is important, especially if the particular region of complementarity in an HSD17B13 gene is known to have polymorphic sequence variation within the population.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA, is un-modified, and does not comprise, e.g., chemical modifications and/or conjugations known in the art and described herein. In another embodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified. iRNAs of the invention in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

In some aspects of the invention, substantially all of the nucleotides of an iRNA of the invention are modified and the iRNA agents comprise no more than 10 nucleotides comprising 2′-fluoro modifications (e.g., no more than 9 2′-fluoro modifications, no more than 8 2′-fluoro modifications, no more than 7 2′-fluoro modifications, no more than 6 2′-fluoro modifications, no more than 5 2′-fluoro modifications, no more than 4 2′-fluoro modifications, no more than 5 2′-fluoro modifications, no more than 4 2′-fluoro modifications, no more than 3 2′-fluoro modifications, or no more than 2 2′-fluoro modifications). For example, in some embodiments, the sense strand comprises no more than 4 nucleotides comprising 2′-fluoro modifications (e.g., no more than 3 2′-fluoro modifications, or no more than 2 2′-fluoro modifications). In other embodiments, the antisense strand comprises no more than 6 nucleotides comprising 2′-fluoro modifications (e.g., no more than 5 2′-fluoro modifications, no more than 4 2′-fluoro modifications, no more than 4 2′-fluoro modifications, or no more than 2 2′-fluoro modifications).

In other aspects of the invention, all of the nucleotides of an iRNA of the invention are modified and the iRNA agents comprise no more than 10 nucleotides comprising 2′-fluoro modifications (e.g., no more than 9 2′-fluoro modifications, no more than 8 2′-fluoro modifications, no more than 7 2′-fluoro modifications, no more than 6 2′-fluoro modifications, no more than 5 2′-fluoro modifications, no more than 4 2′-fluoro modifications, no more than 5 2′-fluoro modifications, no more than 4 2′-fluoro modifications, no more than 3 2′-fluoro modifications, or no more than 2 2′-fluoro modifications).

In one embodiment, the double stranded RNAi agent of the invention further comprises a 5′-phosphate or a 5′-phosphate mimic at the 5′ nucleotide of the antisense strand. In another embodiment, the double stranded RNAi agent further comprises a 5′-phosphate mimic at the 5′ nucleotide of the antisense strand. In a specific embodiment, the 5′-phosphate mimic is a 5′-vinyl phosphate (5′-VP).

The nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. In some embodiments of the invention, the dsRNA agents of the invention are in a free acid form. In other embodiments of the invention, the dsRNA agents of the invention are in a salt form. In one embodiment, the dsRNA agents of the invention are in a sodium salt form. In certain embodiments, when the dsRNA agents of the invention are in the sodium salt form, sodium ions are present in the agent as counterions for substantially all of the phosphodiester and/or phosphorothiotate groups present in the agent. Agents in which substantially all of the phosphodiester and/or phosphorothioate linkages have a sodium counterion include not more than 5, 4, 3, 2, or 1 phosphodiester and/or phosphorothioate linkages without a sodium counterion. In some embodiments, when the dsRNA agents of the invention are in the sodium salt form, sodium ions are present in the agent as counterions for all of the phosphodiester and/or phosphorothiotate groups present in the agent.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_nO]_mCH₃, O(CH₂)._nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁to C₁₀lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include: 5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides, 5′-Me-2′-deoxynucleotides, (both R and S isomers in these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An iRNA of the invention can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

An iRNA of the invention can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

An iRNA of the invention can also be modified to include one or more bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A“bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH2-O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the invention include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

Additional representative U.S. patents and US Patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

An iRNA of the invention can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)-0-2′ bridge. In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”

An iRNA of the invention may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US Patent Publication No. 2013/0190383; and PCT publication WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, an iRNA of the invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).

Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

Other modifications of an iRNA of the invention include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of an RNAi agent. Suitable phosphate mimics are disclosed in, for example US Patent Publication No. 2012/0157511, the entire contents of which are incorporated herein by reference.

In certain specific embodiments, an RNAi agent of the present invention is an agent that inhibits the expression of an HSD17B13 gene which is selected from the group of agents listed in any one of Tables 2, 3, 7, 8, 10, 11, or 13. Any of these agents may further comprise a ligand.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNAi agents of the invention include agents with chemical modifications as disclosed, for example, in WO 2013/075035, filed on Nov. 16, 2012, the entire contents of which are incorporated herein by reference.

Accordingly, the invention provides double stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., an HSD17B13 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may range from 12-30 nucleotides in length. For example, each strand may be between 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. In one embodiment, the sense strand is 21 nucleotides in length. In one embodiment, the antisense strand is 23 nucleotides in length.

The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” The duplex region of an RNAi agent may be 12-30 nucleotide pairs in length. For example, the duplex region can be between 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhang regions and/or capping groups at the 3′-end, 5′-end, or both ends of one or both strands. The overhang can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2-F, 2′-Omethyl, thymidine (T), 2 custom-character -O-methoxyethyl-5-methyluridine (Teo), 2-O-methoxyethyladenosine (Aeo), 2-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3′-end of the sense strand, antisense strand, or both strands. In one embodiment, this 3′-overhang is present in the antisense strand. In one embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3-terminal end of the sense strand or, alternatively, at the 3-terminal end of the antisense strand. The RNAi may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the RNAi has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7, 8, 9 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8, 9, 10 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide overhang. Preferably, the 2 nucleotide overhang is at the 3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand. In one embodiment, every nucleotide in the sense strand and the antisense strand of the RNAi agent, including the nucleotides that are part of the motifs are modified nucleotides. In one embodiment each residue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc₃).

In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3′ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3′ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands, wherein the RNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand and the 5′ end of the second strand form a blunt end and the second strand is 1-4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region region which is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein dicer cleavage of the RNAi agent preferentially results in an siRNA comprising the 3′ end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For an RNAi agent having a duplex region of 17-23 nucleotide in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1^stnucleotide from the 5′-end of the antisense strand, or, the count starting from the 1^stpaired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other then the chemistry of the motifs are distinct from each other and when the motifs are separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent may contain more than one motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end or both ends of the strand.

In another embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two or three nucleotides.

When the sense strand and the antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two or three nucleotides in the duplex region.

In one embodiment, every nucleotide in the sense strand and antisense strand of the RNAi agent, including the nucleotides that are part of the motifs, may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of a RNA or may only occur in a single strand region of a RNA. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. For example, it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In one embodiment, each residue of the sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-hydroxyl, or 2′-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_aand/or N_bcomprise modifications of an alternating pattern. The term “alternating motif” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In one embodiment, the RNAi agent of the invention comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 5′-3′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 5′-3′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

In one embodiment, the RNAi agent comprises the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the sense strand initially has a shift relative to the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the antisense strand initially, i.e., the 2′-O-methyl modified nucleotide on the sense strand base pairs with a 2′-F modified nucleotide on the antisense strand and vice versa. The 1 position of the sense strand may start with the 2′-F modification, and the 1 position of the antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand and/or antisense strand interrupts the initial modification pattern present in the sense strand and/or antisense strand. This interruption of the modification pattern of the sense and/or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense and/or antisense strand surprisingly enhances the gene silencing activity to the target gene.

In one embodiment, when the motif of three identical modifications on three consecutive nucleotides is introduced to any of the strands, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, the portion of the sequence containing the motif is “ . . . N_aYYYN_b. . . ,” where “Y” represents the modification of the motif of three identical modifications on three consecutive nucleotide, and “N_a” and “N_b” represent a modification to the nucleotide next to the motif “YYY” that is different than the modification of Y, and where N_aand N_bcan be the same or different modifications. Alternatively, N_aand/or N_bmay be present or absent when there is a wing modification present.

The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both strands in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand and/or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand and/or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. In one embodiment, a double-stranded RNAi agent comprises 6-8 phosphorothioate internucleotide linkages. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5′-terminus or the 3′-terminus.

In one embodiment, the RNAi comprises a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within the duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. These terminal three nucleotides may be at the 3′-end of the antisense strand, the 3′-end of the sense strand, the 5′-end of the antisense strand, and/or the 5′end of the antisense strand.

In one embodiment, the 2 nucleotide overhang is at the 3′-end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. Optionally, the RNAi agent may additionally have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch may occur in the overhang region or the duplex region. The base pair may be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand independently selected from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

In another embodiment, the nucleotide at the 3′-end of the sense strand is deoxy-thymine (dT). In another embodiment, the nucleotide at the 3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment, there is a short sequence of deoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-end of the sense and/or antisense strand.

In one embodiment, the sense strand sequence may be represented by formula (I):

5′n_p-N_a—(XXX)_i—N_b—YYY—N_b—(ZZZ)_j—N_a-n_q3′ (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

- each N_aindependently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;
- each N_bindependently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;
- each n_pand n_qindependently represent an overhang nucleotide;
- wherein Nb and Y do not have the same modification; and
- XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.

In one embodiment, the N_aand/or N_bcomprise modifications of alternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sense strand, the count starting from the 1^stnucleotide, from the 5′-end; or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

5′n_p-N_a—YYY—N_b—ZZZ—N_a-n_q3′ (Ib);

5′n_p-N_a—XXX—N_b—YYY—N_a-n_q3′ (Ic); or

5′n_p-N_a—XXX—N_b—YYY—N_b—ZZZ—N_a-n_q3′ (Id).

When the sense strand is represented by formula (Ib), N_brepresents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_aindependently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N_brepresents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_acan independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each N_bindependently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_bis 0, 1, 2, 3, 4, 5 or 6. Each N_acan independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:

5′n_p-N_a—YYY—N_a-n_q3′ (Ia).

When the sense strand is represented by formula (Ia), each N_aindependently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II):

5′n_q′-N_a′—(Z′Z′Z′)_k—N_b′—Y′Y′Y′—N_b′—(X′X′X′)_l—N′_a-n_p′3′ (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

- each N_a′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;
- each N_b′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;
- each n_p′ and n_q′ independently represent an overhang nucleotide;
- wherein N_b′ and Y′ do not have the same modification; and
- X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, the N_a′ and/or N_b′ comprise modifications of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotide in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the 1^stnucleotide, from the 5′-end; or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

The antisense strand can therefore be represented by the following formulas:

5′n_q′—N_a′—Z′Z′Z′—N_b′—Y′Y′Y′—N_a′-n_p′3′ (IIb);

5′n_q′-N_a′—Y′Y′Y′—N_b′—X′X′X′-n_p′3′ (IIc); or

5′n_q′-N_a′—Z′Z′Z′—N_b′—Y′Y′Y′—N_b′—X′X′X′—N_a′-n_p′3′ (IId).

When the antisense strand is represented by formula (IIb), N_b′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), N_b′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N_b′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N_bis 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may be represented by the formula:

5′n_p′-N_a′—Y′Y′Y′—N_a′-n_q′3′ (Ia).

When the antisense strand is represented as formula (IIa), each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1^stnucleotide from the 5′-end, or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1st nucleotide from the 5′ end, or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, the RNAi agents for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (III):

sense: 5′np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3′

antisense: 3′np′-Na′-(X′X′X′)k-Nb′-Y′Y′Y′—Nb′-(Z′Z′Z′)l-Na′-nq′5′ (III)

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

- each Na and Na′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;
- each Nb and Nb′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;
- wherein each np′, np, nq′, and nq, each of which may or may not be present, independently represents an overhang nucleotide; and
- XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forming a RNAi duplex include the formulas below:

5′np-Na-YYY-Na-nq3′

3′np′-Na′-Y′Y′Y′—Na′nq′5′ (IIIa)

5′np-Na-YYY-Nb-ZZZ-Na-nq3′

3′np′-Na′-Y′Y′Y′—Nb′-Z′Z′Z′—Na′nq′5′ (IIIb)

5′np-Na-XXX-Nb-YYY-Na-nq3′

3′np′-Na′-X′X′X′—Nb′-Y′Y′Y′—Na′-nq′5′ (IIIc)

5′np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3′

3′np′-Na′-X′X′X′—Nb′-Y′Y′Y′—Nb′-Z′Z′Z′—Na-nq′5′ (IIId)

When the RNAi agent is represented by formula (IIIa), each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each Nb independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each Nb, Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each Nb, Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na, Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na′, Nb and Nb′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications and np′>0 and at least one np′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends and are optionally conjugated to to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

In certain embodiments, an RNAi agent of the invention may contain a low number of nucleotides containing a 2′-fluoro modification, e.g., 10 or fewer nucleotides with 2′-fluoro modification. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleotides with a 2′-fluoro modification. In a specific embodiment, the RNAi agent of the invention contains 10 nucleotides with a 2′-fluoro modification, e.g., 4 nucleotides with a 2′-fluoro modification in the sense strand and 6 nucleotides with a 2′-fluoro modification in the antisense strand. In another specific embodiment, the RNAi agent of the invention contains 6 nucleotides with a 2′-fluoro modification, e.g., 4 nucleotides with a 2′-fluoro modification in the sense strand and 2 nucleotides with a 2′-fluoro modification in the antisense strand.

In other embodiments, an RNAi agent of the invention may contain an ultra low number of nucleotides containing a 2′-fluoro modification, e.g., 2 or fewer nucleotides containing a 2′-fluoro modification. For example, the RNAi agent may contain 2, 1 of 0 nucleotides with a 2′-fluoro modification. In a specific embodiment, the RNAi agent may contain 2 nucleotides with a 2′-fluoro modification, e.g., 0 nucleotides with a 2-fluoro modification in the sense strand and 2 nucleotides with a 2′-fluoro modification in the antisense strand.

Various publications describe multimeric RNAi agents that can be used in the methods of the invention. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520 the entire contents of each of which are hereby incorporated herein by reference.

As described in more detail below, the RNAi agent that contains conjugations of one or more carbohydrate moieties to a RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

In another embodiment of the invention, an iRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The RNAi agent may be represented by formula (L):

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In formula (L), B1, B2, B3, B1′, B2′, B3′, and B4′ each are independently a nucleotide containing a modification selected from the group consisting of 2′-O-alkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA, and BNA/LNA. In certain embodiments, B1, B2, B3, B1′, B2′, B3′, and B4′ each contain 2′-OMe modifications. In certain embodiments, B1, B2, B3, B1′, B2′, B3′, and B4′ each contain 2′-OMe or 2′-F modifications. In certain embodiments, at least one of B1, B2, B3, B1′, B2′, B3′, and B4′ contain 2′-O—N-methylacetamido (2′-O-NMA) modification.

C1 is a thermally destabilizing nucleotide placed at a site opposite to the seed region of the antisense strand (i.e., at positions 2-8 of the 5′-end of the antisense strand). For example, C1 is at a position of the sense strand that pairs with a nucleotide at positions 2-8 of the 5′-end of the antisense strand. In one example, C1 is at position 15 from the 5′-end of the sense strand. C1 nucleotide bears the thermally destabilizing modification which can include abasic modification; mismatch with the opposing nucleotide in the duplex; and sugar modification such as 2′-deoxy modification or acyclic nucleotide e.g., unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA). In certain embodiments, C1 has thermally destabilizing modification selected from the group consisting of: i) mismatch with the opposing nucleotide in the antisense strand; ii) abasic modification selected from the group consisting of:

embedded image

and iii) sugar modification selected from the group consisting of:

embedded image

wherein B is a modified or unmodified nucleobase, R¹and R²independently are H, halogen, OR₃, or alkyl; and R₃is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar. In certain embodiments, the thermally destabilizing modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; and optionally, at least one nucleobase in the mismatch pair is a 2′-deoxy nucleobase. In one example, the thermally destabilizing modification in C1 is GNA or

embedded image

T1, T1′, T2′, and T3′ each independently represent a nucleotide comprising a modification providing the nucleotide a steric bulk that is less or equal to the steric bulk of a 2′-OMe modification. A steric bulk refers to the sum of steric effects of a modification. Methods for determining steric effects of a modification of a nucleotide are known to one skilled in the art. The modification can be at the 2′ position of a ribose sugar of the nucleotide, or a modification to a non-ribose nucleotide, acyclic nucleotide, or the backbone of the nucleotide that is similar or equivalent to the 2′ position of the ribose sugar, and provides the nucleotide a steric bulk that is less than or equal to the steric bulk of a 2′-OMe modification. For example, T1, T1′, T2′, and T3′ are each independently selected from DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl. In certain embodiments, T1 is DNA. In certain embodiments, T1′ is DNA, RNA or LNA. In certain embodiments, T2′ is DNA or RNA. In certain embodiments, T3′ is DNA or RNA.

n¹, n³, and q¹are independently 4 to 15 nucleotides in length.

n⁵, q³, and q⁷are independently 1-6 nucleotide(s) in length.

n⁴, q², and q⁶are independently 1-3 nucleotide(s) in length; alternatively, n⁴is 0.

q⁵is independently 0-10 nucleotide(s) in length.

n²and q⁴are independently 0-3 nucleotide(s) in length.

Alternatively, n⁴is 0-3 nucleotide(s) in length.

In certain embodiments, n⁴can be 0. In one example, n⁴is 0, and q²and q⁶are 1. In another example, n⁴is 0, and q²and q⁶are 1, with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In certain embodiments, n⁴, q², and q⁶are each 1.

In certain embodiments, n², n⁴, q², q⁴, and q⁶are each 1.

In certain embodiments, C1 is at position 14-17 of the 5′-end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n⁴is 1. In certain embodiments, C1 is at position 15 of the 5′-end of the sense strand

In certain embodiments, T3′ starts at position 2 from the 5′ end of the antisense strand. In one example, T3′ is at position 2 from the 5′ end of the antisense strand and q⁶is equal to 1.

In certain embodiments, T1′ starts at position 14 from the 5′ end of the antisense strand. In one example, T1′ is at position 14 from the 5′ end of the antisense strand and q²is equal to 1.

In an exemplary embodiment, T3′ starts from position 2 from the 5′ end of the antisense strand and T1′ starts from position 14 from the 5′ end of the antisense strand. In one example, T3′ starts from position 2 from the 5′ end of the antisense strand and q⁶is equal to 1 and T1′ starts from position 14 from the 5′ end of the antisense strand and q²is equal to 1.

In certain embodiments, T1′ and T3′ are separated by 11 nucleotides in length (i.e. not counting the T1′ and T3′ nucleotides).

In certain embodiments, T1′ is at position 14 from the 5′ end of the antisense strand. In one example, T1′ is at position 14 from the 5′ end of the antisense strand and q²is equal to 1, and the modification at the 2′ position or positions in a non-ribose, acyclic or backbone that provide less steric bulk than a 2′-OMe ribose.

In certain embodiments, T3′ is at position 2 from the 5′ end of the antisense strand. In one example, T3′ is at position 2 from the 5′ end of the antisense strand and q⁶is equal to 1, and the modification at the 2′ position or positions in a non-ribose, acyclic or backbone that provide less than or equal to steric bulk than a 2′-OMe ribose.

In certain embodiments, T1 is at the cleavage site of the sense strand. In one example, T1 is at position 11 from the 5′ end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n²is 1. In an exemplary embodiment, T1 is at the cleavage site of the sense strand at position 11 from the 5′ end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n²is 1,

In certain embodiments, T2′ starts at position 6 from the 5′ end of the antisense strand. In one example, T2′ is at positions 6-10 from the 5′ end of the antisense strand, and q⁴is 1.

In an exemplary embodiment, T1 is at the cleavage site of the sense strand, for instance, at position 11 from the 5′ end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n²is 1; T1′ is at position 14 from the 5′ end of the antisense strand, and q²is equal to 1, and the modification to T1′ is at the 2′ position of a ribose sugar or at positions in a non-ribose, acyclic or backbone that provide less steric bulk than a 2′-OMe ribose; T2′ is at positions 6-10 from the 5′ end of the antisense strand, and q⁴is 1; and T3′ is at position 2 from the 5′ end of the antisense strand, and q⁶is equal to 1, and the modification to T3′ is at the 2′ position or at positions in a non-ribose, acyclic or backbone that provide less than or equal to steric bulk than a 2′-OMe ribose.

In certain embodiments, T2′ starts at position 8 from the 5′ end of the antisense strand. In one example, T2′ starts at position 8 from the 5′ end of the antisense strand, and q⁴is 2.

In certain embodiments, T2′ starts at position 9 from the 5′ end of the antisense strand. In one example, T2′ is at position 9 from the 5′ end of the antisense strand, and q⁴is 1.

In certain embodiments, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 1, B3′ is 2′-OMe or 2′-F, q⁵is 6, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In certain embodiments, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 1, B3′ is 2′-OMe or 2′-F, q⁵is 6, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 6, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 7, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 6, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 7, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 1, B3′ is 2′-OMe or 2′-F, q⁵is 6, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 1, B3′ is 2′-OMe or 2′-F, q⁵is 6, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 5, T2′ is 2′-F, q⁴is 1, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; optionally with at least 2 additional TT at the 3′-end of the antisense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 5, T2′ is 2′-F, q⁴is 1, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; optionally with at least 2 additional TT at the 3′-end of the antisense strand; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

The RNAi agent can comprise a phosphorus-containing group at the 5′-end of the sense strand or antisense strand. The 5′-end phosphorus-containing group can be 5′-end phosphate (5′-P), 5′-end phosphorothioate (5′-PS), 5′-end phosphorodithioate (5′-PS₂), 5′-end vinylphosphonate (5′-VP), 5′-end methylphosphonate (MePhos), or 5′-deoxy-5′-C-malonyl

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When the 5′-end phosphorus-containing group is 5′-end vinylphosphonate (5′-VP), the 5′-VP can be either 5′-E-VP isomer (i.e., trans-vinylphosphate,

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5′-Z-VP isomer (i.e., cis-vinylphosphate,

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or mixtures thereof.

In certain embodiments, the RNAi agent comprises a phosphorus-containing group at the 5′-end of the sense strand. In certain embodiments, the RNAi agent comprises a phosphorus-containing group at the 5′-end of the antisense strand.

In certain embodiments, the RNAi agent comprises a 5′-P. In certain embodiments, the RNAi agent comprises a 5′-P in the antisense strand.

In certain embodiments, the RNAi agent comprises a 5′-PS. In certain embodiments, the RNAi agent comprises a 5′-PS in the antisense strand.

In certain embodiments, the RNAi agent comprises a 5′-VP. In certain embodiments, the RNAi agent comprises a 5′-VP in the antisense strand. In certain embodiments, the RNAi agent comprises a 5′-E-VP in the antisense strand. In certain embodiments, the RNAi agent comprises a 5′-Z-VP in the antisense strand.

In certain embodiments, the RNAi agent comprises a 5′-PS₂. In certain embodiments, the RNAi agent comprises a 5′-PS₂in the antisense strand.

In certain embodiments, the RNAi agent comprises a 5′-PS₂. In certain embodiments, the RNAi agent comprises a 5′-deoxy-5′-C-malonyl in the antisense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1. The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-P.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-PS.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-PS₂.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1. The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P and a targeting ligand. In certain embodiments, the 5′-P is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS and a targeting ligand. In certain embodiments, the 5′-PS is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, or combination thereof), and a targeting ligand.

In certain embodiments, the 5′-VP is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂and a targeting ligand. In certain embodiments, the 5′-PS₂is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targeting ligand. In certain embodiments, the 5′-deoxy-5′-C-malonyl is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-P and a targeting ligand. In certain embodiments, the 5′-P is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-PS and a targeting ligand. In certain embodiments, the 5′-PS is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, or combination thereof) and a targeting ligand. In certain embodiments, the 5′-VP is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-PS₂and a targeting ligand. In certain embodiments, the 5′-PS₂is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-OMe, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targeting ligand. In certain embodiments, the 5′-deoxy-5′-C-malonyl is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P and a targeting ligand. In certain embodiments, the 5′-P is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS and a targeting ligand. In certain embodiments, the 5′-PS is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, or combination thereof) and a targeting ligand. In certain embodiments, the 5′-VP is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂and a targeting ligand. In certain embodiments, the 5′-PS₂is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, T2′ is 2′-F, q⁴is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targeting ligand. In certain embodiments, the 5′-deoxy-5′-C-malonyl is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P and a targeting ligand. In certain embodiments, the 5′-P is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS and a targeting ligand. In certain embodiments, the 5′-PS is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, or combination thereof) and a targeting ligand. In certain embodiments, the 5′-VP is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂and a targeting ligand. In certain embodiments, the 5′-PS₂is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In certain embodiments, B1 is 2′-OMe or 2′-F, n¹is 8, T1 is 2′F, n²is 3, B2 is 2′-OMe, n³is 7, n⁴is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹is 9, T1′ is 2′-F, q²is 1, B2′ is 2′-OMe or 2′-F, q³is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵is 7, T3′ is 2′-F, q⁶is 1, B4′ is 2′-F, and q⁷is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targeting ligand. In certain embodiments, the 5′-deoxy-5′-C-malonyl is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In a particular embodiment, an RNAi agent of the present invention comprises:

(a) a sense strand having:

- (i) a length of 21 nucleotides;
- (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; and
- (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21, and 2′-OMe modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5′ end);

and

(b) an antisense strand having:

- (i) a length of 23 nucleotides;
- (ii) 2′-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21, and 23, and 2′F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the 5′ end); and
- (iii) phosphorothioate internucleotide linkages between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end);

wherein the dsRNA agents have a two-nucleotide overhang at the 3′-end of the antisense strand, and a blunt end at the 5′-end of the antisense strand.

In another particular embodiment, an RNAi agent of the present invention comprises:

(a) a sense strand having:

- (i) a length of 21 nucleotides;
- (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker;
- (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19, and 21, and 2′-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5′ end); and
- (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

- (i) a length of 23 nucleotides;
- (ii) 2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2′F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5′ end); and
- (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end);

wherein the RNAi agents have a two-nucleotide overhang at the 3′-end of the antisense strand, and a blunt end at the 5′-end of the antisense strand.

In another particular embodiment, an RNAi agent of the present invention comprises:

(a) a sense strand having:

- (i) a length of 21 nucleotides;
- (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker;
- (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2′-F modifications at positions 7, and 9, and a desoxy-nucleotide (e.g. dT) at position 11 (counting from the 5′ end); and
- (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

- (i) a length of 23 nucleotides;
- (ii) 2′-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19 to 23, and 2′-F modifications at positions 2, 4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5′ end); and
- (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end);

wherein the RNAi agents have a two-nucleotide overhang at the 3′-end of the antisense strand, and a blunt end at the 5′-end of the antisense strand.

In another particular embodiment, an RNAi agent of the present invention comprises:

(a) a sense strand having:

- (i) a length of 21 nucleotides;
- (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker;
- (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2′-F modifications at positions 7, 9, 11, 13, and 15; and
- (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

- (i) a length of 23 nucleotides;
- (ii) 2′-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21 to 23, and 2′-F modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5′ end); and
- (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end);

wherein the RNAi agents have a two-nucleotide overhang at the 3′-end of the antisense strand, and a blunt end at the 5′-end of the antisense strand.

In another particular embodiment, an RNAi agent of the present invention comprises:

(a) a sense strand having:

- (i) a length of 21 nucleotides;
- (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker;
- (iii) 2′-OMe modifications at positions 1 to 9, and 12 to 21, and 2′-F modifications at positions 10, and 11; and
- (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

- (i) a length of 23 nucleotides;
- (ii) 2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2′-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5′ end); and
- (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end);

wherein the RNAi agents have a two-nucleotide overhang at the 3′-end of the antisense strand, and a blunt end at the 5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

(a) a sense strand having:

- (i) a length of 21 nucleotides;
- (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker;
- (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11, and 13, and 2′-OMe modifications at positions 2, 4, 6, 8, 12, and 14 to 21; and
- (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

- (i) a length of 23 nucleotides;
- (ii) 2′-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19, and 21 to 23, and 2′-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5′ end); and
- (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end);

wherein the RNAi agents have a two-nucleotide overhang at the 3′-end of the antisense strand, and a blunt end at the 5′-end of the antisense strand.

In another particular embodiment, an RNAi agent of the present invention comprises:

(a) a sense strand having:

- (i) a length of 21 nucleotides;
- (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker;
- (iii) 2′-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19 to 21, and 2′-F modifications at positions 3, 5, 7, 9 to 11, 13, 16, and 18; and
- (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

- (i) a length of 25 nucleotides;
- (ii) 2′-OMe modifications at positions 1, 4, 6, 7, 9, 11 to 13, 15, 17, and 19 to 23, 2′-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and desoxy-nucleotides (e.g. dT) at positions 24 and 25 (counting from the 5′ end); and
- (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end);

wherein the RNAi agents have a four-nucleotide overhang at the 3′-end of the antisense strand, and a blunt end at the 5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

(a) a sense strand having:

- (i) a length of 21 nucleotides;
- (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker;
- (iii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2′-F modifications at positions 7, and 9 to 11; and
- (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

- (i) a length of 23 nucleotides;
- (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15, and 17 to 23, and 2′-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5′ end); and
- (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end);

wherein the RNAi agents have a two-nucleotide overhang at the 3′-end of the antisense strand, and a blunt end at the 5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

(a) a sense strand having:

- (i) a length of 21 nucleotides;
- (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker;
- (iii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2′-F modifications at positions 7, and 9 to 11; and
- (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

- (i) a length of 23 nucleotides;
- (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23, and 2′-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5′ end); and
- (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end);

wherein the RNAi agents have a two-nucleotide overhang at the 3′-end of the antisense strand, and a blunt end at the 5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

(a) a sense strand having:

- (i) a length of 19 nucleotides;
- (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker;
- (iii) 2′-OMe modifications at positions 1 to 4, 6, and 10 to 19, and 2′-F modifications at positions 5, and 7 to 9; and
- (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

- (i) a length of 21 nucleotides;
- (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 21, and 2′-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5′ end); and
- (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5′ end);

wherein the RNAi agents have a two-nucleotide overhang at the 3′-end of the antisense strand, and a blunt end at the 5′-end of the antisense strand.

In certain embodiments, the iRNA for use in the methods of the invention is an agent selected from agents listed in Tables 2, 3, 7, 8, 10, 11, or 13. In one embodiment, the agent is AD-288917. In another embodiment, the agent is AD-288996. In another embodiment, the agent is AD-413639. In one embodiment, the agent is AD-413644. In another embodiment, the agent is AD-413669. These agents may further comprise a ligand.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556), cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 2977). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:2978) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 2979) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 2980) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidomimetics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

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wherein Y is O or S and n is 3-6 (Formula XXV);

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wherein X is O or S (Formula XXVII);

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In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as

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Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

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when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent, e.g., the 3′ or 5′end of the sense strand of a dsRNA agent as described herein. In another embodiment, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) of GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

Additional carbohydrate conjugates (and linkers) suitable for use in the present invention include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

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when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention, a ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XLV)-(XLVI):

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wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

P^2A, P^2B, P^3A, P^3B, P^4A, P^4B, P^5A, P^5B, P^5C, T^2A, T^2B, T^3A, T^3B, T^4A, T^4B, T^4A, T^5B, T^5Care each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q^2A, Q^2B, Q^3A, Q^3B, Q^4A, Q^4B, Q^5A, Q^5B, Q^5Care independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^N), C(R′)═C(R″), C≡C or C(O);

R^2A, R^2B, R^3A, R^3B, R^4A, R^4B, R^5A, R^5B, R^5Care each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^a)C(O), —C(O)—CH(R^a)—NH—, CO, CH═N—O,

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or heterocyclyl;

L^2A, L^2B, L^3A, L^3B, L^4A, L^4B, L^5A, L^5Band L^5Crepresent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^ais H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XLIX):

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- wherein L^5A, L^5Band L^5Crepresent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a disorder of lipid metabolism) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J. et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J. et al. (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et a.l (2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience 129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O. et al., (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H. et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the HSD17B13 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. Accordingly, in one embodiment, provided herein are pharmaceutical compositions comprising a double stranded ribonucleic acid (dsRNA) agent that inhibits expression of 17β-hydroxysteroid dehydrogenases type 13 (HSD17B13) in a cell, such as a liver cell, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and said antisense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:7; and a pharmaceutically acceptable carrier. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of SEQ ID NO:1, and said antisense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of SEQ ID NO:7.

In another embodiment, provided herein are pharmaceutical compositions comprising a dsRNA agent that inhibits expression of 17β-Hydroxysteroid dehydrogenases (HSD17B13) in a cell, such as a liver cell, wherein the dsRNA agent comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from any one of the antisense sequences listed in any one of Tables 2, 3, 7, 8, 10, 11, or 13; and a pharmaceutically acceptable carrier. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides from any one of the antisense sequences listed in any one of Tables 2, 3, 7, 8, 10, 11, or 13.

The pharmaceutical compositions containing the iRNA of the invention are useful for treating a disease or disorder associated with the expression or activity of an HSD17B13 gene, e.g., a chronic fibro-inflammatory disease.

Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV), intramuscular (IM) or for subcutaneous delivery. Another example is compositions that are formulated for direct delivery into the liver, e.g., by infusion into the liver, such as by continuous pump infusion. The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of an HSD17B13 gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. Typically, a suitable dose of an iRNA of the invention will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg.

A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day to once a year. In certain embodiments, the iRNA is administered about once per week, once every 7-10 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once per month, once every 2 months, once every 3 months (once per quarter), once every 4 months, once every 5 months, or once every 6 months.

After an initial treatment regimen, the treatments can be administered on a less frequent basis.

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as an HSD17B13-associated disease, disorder, or condition that would benefit from reduction in the expression of HSD17B13. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose. Suitable mouse models are known in the art and include, for example, mice and rats fed a high fat diet (HFD; also referred to as a Western diet), a methionine-choline deficient (MCD) diet, or a high-fat (15%), high-cholesterol (1%) diet (HFHC), an obese (ob/ob) mouse containing a mutation in the obese (ob) gene (Wiegman et al., (2003) Diabetes, 52:1081-1089); a mouse containing homozygous knock-out of an LDL receptor (LDLR−/− mouse; Ishibashi et al., (1993) J Clin Invest 92(2):883-893); diet-induced artherosclerosis mouse model (Ishida et al., (1991) J. Lipid. Res., 32:559-568); heterozygous lipoprotein lipase knockout mouse model (Weistock et al., (1995) J. Clin. Invest. 96(6):2555-2568); mice and rats fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) (Matsumoto et al. (2013) Int. J. Exp. Path. 94:93-103); mice and rats fed a high-trans-fat, cholesterol diet (HTF-C) (Clapper et al. (2013) Am. J. Physiol. Gastrointest. Liver Physiol. 305:G483-G495); mice and rats fed a high-fat, high-cholesterol, bile salt diet (HF/HC/BS) (Matsuzawa et al. (2007) Hepatology 46:1392-1403); and mice and rats fed a high-fat diet+fructose (30%) water (Softic et al. (2018) J. Clin. Invest. 128(1)-85-96).

The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.

The iRNA can be delivered in a manner to target a particular cell or tissue, such as the liver (e.g., the hepatocytes of the liver).

In some embodiments, the pharmaceutical compositions of the invention are suitable for intramuscular administration to a subject. In other embodiments, the pharmaceutical compositions of the invention are suitable for intravenous administration to a subject. In some embodiments of the invention, the pharmaceutical compositions of the invention are suitable for subcutaneous administration to a subject, e.g., using a 29 g or 30 g needle.

The pharmaceutical compositions of the invention may include an RNAi agent of the invention in an unbuffered solution, such as saline or water, or in a buffer solution, such as a buffer solution comprising acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.

In one embodiment, the pharmaceutical compositions of the invention, e.g., such as the compositions suitable for subcutaneous administration, comprise an RNAi agent of the invention in phosphate buffered saline (PBS). Suitable concentrations of PBS include, for example, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 6.5 mM, 7 mM, 7.5.mM, 9 mM, 8.5 mM, 9 mM, 9.5 mM, or about 10 mM PBS. In one embodiment of the invention, a pharmaceutical composition of the invention comprises an RNAi agent of the invention dissolved in a solution of about 5 mM PBS (e.g., 0.64 mM NaH₂PO₄, 4.36 mM Na₂HPO₄, 85 mM NaCl). Values intermediate to the above recited ranges and values are also intended to be part of this invention. In addition, ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.

The pH of the pharmaceutical compositions of the invention may be between about 5.0 to about 8.0, about 5.5 to about 8.0, about 6.0 to about 8.0, about 6.5 to about 8.0, about 7.0 to about 8.0, about 5.0 to about 7.5, about 5.5 to about 7.5, about 6.0 to about 7.5, about 6.5 to about 7.5, about 5.0 to about 7.2, about 5.25 to about 7.2, about 5.5 to about 7.2, about 5.75 to about 7.2, about 6.0 to about 7.2, about 6.5 to about 7.2, or about 6.8 to about 7.2. Ranges and values intermediate to the above recited ranges and values are also intended to be part of this invention.

The osmolality of the pharmaceutical compositions of the invention may be suitable for subcutaneous administration, such as no more than about 400 mOsm/kg, e.g., between 50 and 400 mOsm/kg, between 75 and 400 mOsm/kg, between 100 and 400 mOsm/kg, between 125 and 400 mOsm/kg, between 150 and 400 mOsm/kg, between 175 and 400 mOsm/kg, between 200 and 400 mOsm/kg, between 250 and 400 mOsm/kg, between 300 and 400 mOsm/kg, between 50 and 375 mOsm/kg, between 75 and 375 mOsm/kg, between 100 and 375 mOsm/kg, between 125 and 375 mOsm/kg, between 150 and 375 mOsm/kg, between 175 and 375 mOsm/kg, between 200 and 375 mOsm/kg, between 250 and 375 mOsm/kg, between 300 and 375 mOsm/kg, between 50 and 350 mOsm/kg, between 75 and 350 mOsm/kg, between 100 and 350 mOsm/kg, between 125 and 350 mOsm/kg, between 150 and 350 mOsm/kg, between 175 and 350 mOsm/kg, between 200 and 350 mOsm/kg, between 250 and 350 mOsm/kg, between 50 and 325 mOsm/kg, between 75 and 325 mOsm/kg, between 100 and 325 mOsm/kg, between 125 and 325 mOsm/kg, between 150 and 325 mOsm/kg, between 175 and 325 mOsm/kg, between 200 and 325 mOsm/kg, between 250 and 325 mOsm/kg, between 300 and 325 mOsm/kg, between 300 and 350 mOsm/kg, between 50 and 300 mOsm/kg, between 75 and 300 mOsm/kg, between 100 and 300 mOsm/kg, between 125 and 300 mOsm/kg, between 150 and 300 mOsm/kg, between 175 and 300 mOsm/kg, between 200 and 300 mOsm/kg, between 250 and 300, between 50 and 250 mOsm/kg, between 75 and 250 mOsm/kg, between 100 and 250 mOsm/kg, between 125 and 250 mOsm/kg, between 150 and 250 mOsm/kg, between 175 and 350 mOsm/kg, between 200 and 250 mOsm/kg, e.g., about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 295, 300, 305, 310, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, or about 400 mOsm/kg. Ranges and values intermediate to the above recited ranges and values are also intended to be part of this invention.

The pharmaceutical compositions of the invention comprising the RNAi agents of the invention, may be present in a vial that contains about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0 mL of the pharmaceutical composition. The concentration of the RNAi agents in the pharmaceutical compositions of the invention may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 130, 125, 130, 135, 140, 145, 150, 175, 180, 185, 190, 195, 200, 205, 210, 215, 230, 225, 230, 235, 240, 245, 250, 275, 280, 285, 290, 295, 300, 305, 310, 315, 330, 325, 330, 335, 340, 345, 350, 375, 380, 385, 390, 395, 400, 405, 410, 415, 430, 425, 430, 435, 440, 445, 450, 475, 480, 485, 490, 495, or about 500 mg/mL. In one embodiment, the concentration of the RNAi agents in the pharmaceutical compositions of the invention is about 100 mg/mL. Values intermediate to the above recited ranges and values are also intended to be part of this invention.

The pharmaceutical compositions of the invention may comprise a dsRNA agent of the invention in a free acid form. In other embodiments of the invention, the pharmaceutical compositions of the invention may comprise a dsRNA agent of the invention in a salt form, such as a sodium salt form. In certain embodiments, when the dsRNA agents of the invention are in the sodium salt form, sodium ions are present in the agent as counterions for substantially all of the phosphodiester and/or phosphorothiotate groups present in the agent. Agents in which substantially all of the phosphodiester and/or phosphorothioate linkages have a sodium counterion include not more than 5, 4, 3, 2, or 1 phosphodiester and/or phosphorothioate linkages without a sodium counterion. In some embodiments, when the dsRNA agents of the invention are in the sodium salt form, sodium ions are present in the agent as counterions for all of the phosphodiester and/or phosphorothiotate groups present in the agent.

Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C_1-20alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes include unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition (e.g., iRNA) to be delivered. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.

A liposome containing an iRNA agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNA agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the iRNA agent and condense around the iRNA agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of iRNA agent.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also be adjusted to favor condensation.

Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA agent preparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185 and 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_M1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_M1or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In some embodiments, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA agent (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA agent into the skin. In some implementations, liposomes are used for delivering iRNA agent to epidermal cells and also to enhance the penetration of iRNA agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276.1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNAs can be delivered, for example, subcutaneously by infection in order to deliver iRNAs to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described in WO 2008/042973.

Transfersomes are yet another type of liposomes and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of iRNA, an alkali metal C₈to C₂₂alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the RNAi and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the RNAi, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNA agents of in the invention may be fully encapsulated in a lipid formulation, e.g., an LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In certain embodiments, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In certain embodiments, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

In certain embodiments, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

LNP01

In certain embodiments, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (e.g., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

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LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are provided in the following table.

TABLE A

Exemplary lipid formulations

cationic lipid/non-cationic lipid/

cholesterol/PEG-lipid conjugate

Cationic Lipid
Lipid:siRNA ratio

SNALP
1,2-Dilinolenyloxy-N,N-
DLinDMA/DPPC/Cholesterol/PEG-cDMA

dimethylaminopropane (DLinDMA)
(57.1/7.1/34.4/1.4)

lipid:siRNA ~7:1

S-XTC
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DPPC/Cholesterol/PEG-cDMA

[1,3]-dioxolane (XTC)
57.1/7.1/34.4/1.4

lipid:siRNA ~7:1

LNP05
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG

[1,3]-dioxolane (XTC)
57.5/7.5/31.5/3.5

lipid:siRNA ~6:1

LNP06
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG

[1,3]-dioxolane (XTC)
57.5/7.5/31.5/3.5

lipid:siRNA ~11:1

LNP07
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG

[1,3]-dioxolane (XTC)
60/7.5/31/1.5,

lipid:siRNA ~6:1

LNP08
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG

[1,3]-dioxolane (XTC)
60/7.5/31/1.5,

lipid:siRNA ~11:1

LNP09
2.2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG

[1,3]-dioxolane (XTC)
50/10/38.5/1.5

Lipid:siRNA 10:1

LNP10
(3aR,5s,6aS)-N,N-dimethyl-2,2-
ALN100/DSPC/Cholesterol/PEG-DMG

di((9Z,12Z)-octadeca-9,12-
50/10/38.5/1.5

dienyl)tetrahydro-3aH-
Lipid:siRNA 10:1

cyclopenta[d][1,3]dioxol-5-amine

(ALN100)

LNP11
(6Z,9Z,28Z,31Z)-heptatriaconta-
MC-3/DSPC/Cholesterol/PEG-DMG

6,9,28,31-tetraen-19-yl 4-
50/10/38.5/1.5

(dimethylamino)butanoate (MC3)
Lipid:siRNA 10:1

LNP12
1,1′-(2-(4-(2-((2-(bis(2-
C12-200/DSPC/Cholesterol/PEG-DMG

hydroxydodecyl)amino)ethyl)(2-
50/10/38.5/1.5

hydroxydodecyl)amino)ethyl)piperazin-
Lipid:siRNA 10:1

1-yl)ethylazanediyl)didodecan-2-ol

(C12-200)

LNP13
XTC
XTC/DSPC/Chol/PEG-DMG

50/10/38.5/1.5

Lipid:siRNA: 33:1

LNP14
MC3
MC3/DSPC/Chol/PEG-DMG

40/15/40/5

Lipid:siRNA: 11:1

LNP15
MC3
MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG

50/10/35/4.5/0.5

Lipid:siRNA: 11:1

LNP16
MC3
MC3/DSPC/Chol/PEG-DMG

50/10/38.5/1.5

Lipid:siRNA: 7:1

LNP17
MC3
MC3/DSPC/Chol/PEG-DSG

50/10/38.5/1.5

Lipid:siRNA: 10:1

LNP18
MC3
MC3/DSPC/Chol/PEG-DMG

50/10/38.5/1.5

Lipid:siRNA: 12:1

LNP19
MC3
MC3/DSPC/Chol/PEG-DMG

50/10/35/5

Lipid:siRNA: 8:1

LNP20
MC3
MC3/DSPC/Chol/PEG-DPG

50/10/38.5/1.5

Lipid:siRNA: 10:1

LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG

50/10/38.5/1.5

Lipid:siRNA: 7:1

LNP22
XTC
XTC/DSPC/Chol/PEG-DSG

50/10/38.5/1.5

Lipid:siRNA: 10:1

DSPC: distearoylphosphatidylcholine

DPPC: dipalmitoylphosphatidylcholine

PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)

PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)

PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.

XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. 61/185,712, filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and International Application No. PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publication. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C_1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0445131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0496813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.). Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

iii. Microparticles

An RNAi agent of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C_1-20alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^aD1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.

Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

vii. Other Components

The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating an HSD17B13-associated disease, disorder, or condition. Examples of such agents include, but are not limited to

pyridoxine, an ACE inhibitor (angiotensin converting enzyme inhibitors), e.g., benazepril (Lotensin); an angiotensin II receptor antagonist (ARB) (e.g., losartan potassium, such as Merck & Co.'s Cozaar®), e.g., Candesartan (Atacand); an HMG-CoA reductase inhibitor (e.g., a statin); calcium binding agents, e.g., Sodium cellulose phosphate (Calcibind); diuretics, e.g., thiazide diuretics, such as hydrochlorothiazide (Microzide); an insulin sensitizer, such as the PPARγ agonist pioglitazone, a glp-1r agonist, such as liraglutatide, vitamin E, an SGLT2 inhibitor, a DPPIV inhibitor, and kidney/liver transplant; or a combination of any of the foregoing.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀(the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED₅₀with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC₅₀(i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by HSD17B13 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Synthesis of Cationic Lipids:

Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles featured in the invention may be prepared by known organic synthesis techniques. All substituents are as defined below unless indicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —OR^x, —NR^xR^y, —NR^xC(═O)R_y, —NR^xSO₂R^y, —C(═O)R^x, —C(═O)OR^x, —C(═O)NR^xR^y, —SO_nR^xand —SO_nNR^xR^y, wherein n is 0, 1 or 2, R^xand R^yare the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —OR^x, heterocycle, —NR^xR^y, —NR^xC(═O)R^y, —NR^xSO₂R^y, —C(═O)R^x, —C(═O)OR^x, —C(═O)NR^xR^y, —SO_nR^xand —SO_nNR^xR^y.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods featured in the invention may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

Synthesis of Formula A:

In certain embodiments, nucleic acid-lipid particles featured in the invention are formulated using a cationic lipid of formula A:

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where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.

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Lipid A, where R₁and R₂are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R₃and R₄are independently lower alkyl or R₃and R₄can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.

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Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3:

Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).

Synthesis of ALNY-100:

Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:

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Synthesis of 515:

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516:

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO3 solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H] −232.3 (96.94%).

Synthesis of 517A and 517B:

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO₃(1×50 mL) solution, water (1×30 mL) and finally with brine (1×50 mL). Organic phase was dried over Na₂SO₄and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude 517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS-[M+H]-266.3, [M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518:

Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na₂SO₄then filtered through Celite® and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.

Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as RiboGreen® (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

VI. Methods of the Invention

The present invention also provides methods of using an iRNA of the invention and/or a composition of the invention to reduce and/or inhibit HSD17B13 expression in a cell, such as a cell in a subject, e.g., a hepatocyte. The methods include contacting the cell with an RNAi agent or pharmaceutical composition comprising an iRNA agent of the invention. In some embodiments, the cell is maintained for a time sufficient to obtain degradation of the mRNA transcript of an HSD17B13 gene.

The present invention also provides methods of using an iRNA of the invention and/or a composition of the invention and an iRNA agent targeting a Patatin-like Phospholipase Domain Containing 3 (PNPLA3) gene and/or pharmaceutical composition comprising an iRNA agent targeting PNPLA3 to reduce and/or inhibit HSD17B13 expression in a cell, such as a cell in a subject, e.g., a hepatocyte.

In addition, the present invention provides methods of inhibiting the accumulation and/or expansion of lipid droplets in a cell, such as a cell in a subject, e.g., a hepatocyte. The methods include contacting the cell with an RNAi agent or pharmaceutical composition comprising an iRNA agent of the invention and an iRNA agent targeting a PNPLA3 gene and/or pharmaceutical composition comprising an iRNA agent targeting PNPLA3. In some embodiments, the cell is maintained for a time sufficient to obtain degradation of the mRNA transcript of an HSD17B13 gene and a PNPLA3 gene.

Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of HSD17B13 may be determined by determining the mRNA expression level of HSD17B13 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR; by determining the protein level of HSD17B13 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques. A reduction in the expression of HSD17B13 may also be assessed indirectly by measuring a decrease in biological activity of HSD17B13, e.g., a decrease in the enzymatic activity of HSD17B13 and/or a decrease in one or more of a lipid, a triglyceride, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C and total cholesterol), or free fatty acids in a plasma, or a tissue sample, and/or a reduction in accumulation of fat and/or expansion of lipid droplets in the liver.

Suitable agents targeting a PNPLA3 gene are described in, for example, U.S. Patent Publication No.: 2017/0340661, the entire contents of which are incorporated herein by reference.

In the methods of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may be any cell that expresses an HSD17B13 gene (and, in some embodiments, a PNPLA3 gene). A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.

HSD17B13 expression is inhibited in the cell by at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%. In preferred embodiments, HSD17B13 expression is inhibited by at least 20%.

In some embodiment, PNPLA3 expression is also inhibited in the cell by at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%. In preferred embodiments, PNPLA3 expression is inhibited by at least 20%.

In one embodiment, the in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the HSD17B13 gene of the mammal to be treated.

In another embodiment, the in vivo methods of the invention may include administering to a subject a composition containing a first iRNA agent and a second iRNA agent, where the first iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the HSD17B13 gene of the mammal to be treated and and the second iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the PNPLA3 gene of the mammal to be treated.

When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of HSD17B13, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

An iRNA of the invention may be present in a pharmaceutical composition, such as in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods for inhibiting the expression of an HSD17B13 gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets an HSD17B13 gene in a cell of the mammal, thereby inhibiting expression of the HSD17B13 gene in the cell.

In some embodiment, the methods include administering to the mammal a composition comprising a dsRNA that targets an HSD17B13 gene in a cell of the mammal, thereby inhibiting expression of the HSD17B13 gene in the cell. In another embodiment, the methods include administering to the mammal a pharmaceutical composition comprising a dsRNA agent that targets an HSD17B13 gene in a cell of the mammal.

In another aspect, the present invention provides use of an iRNA agent or a pharmaceutical composition of the invention for inhibiting the expression of an HSD17B13 gene in a mammal.

In yet another aspect, the present invention provides use of an iRNA agent of the invention targeting a HSD17B13 gene or a pharmaceutical composition comprising such an agent in the manufacture of a medicament for inhibiting expression of a HSD17B13 gene in a mammal.

In another aspect, the present invention also provides methods for inhibiting the expression of an HSD17B13 gene and a PNPLA3 gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets an HSD17B13 gene in a cell of the mammal and a composition comprising a dsRNA that targets an PNPLA3 gene in a cell of the mammal, thereby inhibiting expression of the HSD17B13 gene and the PNPLA3 gene in the cell. In one embodiment, the methods include administering to the mammal a pharmaceutical composition comprising a dsRNA agent that targets an HSD17B13 gene and a PNPLA3 gene in a cell of the mammal.

In one aspect, the present invention provides use of an iRNA agent or a pharmaceutical composition of the invention, and a dsRNA that targets a PNPLA3 gene or a pharmaceutical composition comprising such an agent for inhibiting the expression of an HSD17B13 gene and a PNPLA3 gene in a mammal.

In yet another aspect, the present invention provides use of an iRNA agent of the invention targeting a HSD17B13 gene or a pharmaceutical composition comprising such an agent, and a dsRNA that targets an PNPLA3 gene or a pharmaceutical composition comprising such an agent in the manufacture of a medicament for inhibiting expression of a HSD17B13 gene and a PNPLA3 gene in a mammal.

Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, enzymatic activity, described herein.

The present invention also provides therapeutic and prophylactic methods which include administering to a subject having, or prone to developing a fatty liver-associated disease, disorder, or condition, the iRNA agents, pharmaceutical compositions comprising an iRNA agent, or vectors comprising an iRNA of the invention.

In one aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in HSD17B13 expression, e.g., an HSD17B13-associated disease.

The treatment methods (and uses) of the invention include administering to the subject, e.g., a human, a therapeutically effective amount of a dsRNA agent that inhibits expression of HSD17B13 or a pharmaceutical composition comprising a dsRNA that inhibits expression of HSD17B13, thereby treating the subject.

In one embodiment, an HSD17B13-associated disease, disorder, or condition is a chronic fibro-inflammatory liver disease. Non-limiting examples of chronic fibro-inflammatory liver diseases include cancer, e.g., cancer, e.g., hepatocellular carcinoma, nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, and nonalcoholic fatty liver disease (NAFLD).

The present invention also provides use of a therapeutically effective amount of an iRNA agent of the invention or a pharmaceutical composition comprising a dsRNA that inhibits expression of HSD17B13 for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of HSD17B13 expression, e.g., an HSD17B13-associated disease, e.g., a chronic fibro-inflammatory disease.

In another aspect, the present invention provides use of an iRNA agent, e.g., a dsRNA, of the invention targeting a HSD17B13 for gene or a pharmaceutical composition comprising an iRNA agent targeting a HSD17B13 for gene in the manufacture of a medicament for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of HSD17B13 for expression, e.g., a HSD17B13-associated disease.

The present invention also provides use of a prophylactically effective amount of an iRNA agent of the invention or a pharmaceutical composition comprising a dsRNA that inhibits expression of HSD17B13 for preventing at least one symptom in a subject having a disorder that would benefit from reduction in HSD17B13 expression, e.g., a chronic fibro-inflammatory disease.

In another aspect, the present invention provides use of an iRNA agent, e.g., a dsRNA, of the invention targeting a HSD17B13 gene or a pharmaceutical composition comprising an iRNA agent targeting a HSD17B13 gene in the manufacture of a medicament for preventing at least one symptom in a subject having a disorder that would benefit from reduction in HSD17B13 expression, e.g., a chronic fibro-inflammatory disease.

In one aspect, the present invention also provides use of a therapeutically effective amount of an iRNA agent of the invention or a pharmaceutical composition comprising a dsRNA that inhibits expression of HSD17B13 in combination with a dsRNA that targets a PNPLA3 gene or a pharmaceutical composition comprising such an agent for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of HSD17B13 expression, e.g., an HSD17B13-associated disease, e.g., a chronic fibro-inflammatory disease.

In one aspect, the present invention also provides use of an iRNA agent, e.g., a dsRNA, of the invention targeting a HSD17B13 gene or a pharmaceutical composition comprising an iRNA agent targeting a HSD17B13 gene in combination with a dsRNA that targets a PNPLA3 gene or a pharmaceutical composition comprising such an agent for preventing at least one symptom in a subject having a disorder that would benefit from reduction in HSD17B13 expression, e.g., a chronic fibro-inflammatory disease.

The combination methods of the invention for treating a subject, e.g., a human subject, having a HSD17B13-associated disease, disorder, or condition, such as a chronic fibro-inflammatory liver disease, e.g., NASH, are useful for treating such subjects as silencing of PNPLA3 decreases steatosis (i.e. liver fat) while silencing HSD17B13 decreases inflammation and fibrosis. For example, genome wide association studies have demonstrated that silencing PNPLA3 and HSD17B13 have an additive effect to decrease NASH pathology. Indeed, a protective loss-of-function HSD17B13 allele was found to be associated with lower prevalence of NASH in subjects with pathogenic PNPLA3 alleles. In subjects having wild-type PNPLA3 alleles which have lower risk of NASH, the added presence of loss-of-function HSD17B13 alleles conferred even greater protection.

Accordingly, in one aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in HSD17B13 expression, e.g., an HSD17B13-associated disease, such as a chronic fibro-inflammatory liver disease (e.g., cancer, e.g., hepatocellular carcinoma, nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, and nonalcoholic fatty liver disease (NAFLD). In one embodiment, the chronic fibro-inflammatory liver disease is NASH.

The combination treatment methods (and uses) of the invention include administering to the subject, e.g., a human subject, a therapeutically effective amount of a dsRNA agent that inhibits expression of HSD17B13 or a pharmaceutical composition comprising a dsRNA that inhibits expression of HSD17B13, and a dsRNA agent that inhibits expression of PNPLA3 or a pharmaceutical composition comprising a dsRNA that inhibits expression of PNPLA3, thereby treating the subject.

In one aspect, the invention provides methods of preventing at least one symptom in a subject having a disorder that would benefit from reduction in HSD17B13 expression, e.g., a chronic fibro-inflammatory disease, e.g., NASH. The methods include administering to the subject a prophylactically effective amount of dsRNA agent or a pharmaceutical composition comprising a dsRNA that inhibits expression of HSD17B13, and a dsRNA agent that inhibits expression of PNPLA3 or a pharmaceutical composition comprising a dsRNA that inhibits expression of PNPLA3, thereby preventing at least one symptom in the subject.

In certain embodiments of the invention the methods may include identifying a subject that would benefit from reduction in HSD17B13 expression. The methods generally include determining whether or not a sample from the subject comprises a nucleic acid encoding a PNPLA3Ile148Met variant or a PNPLA3Ile144Met variant. The methods may also include classifying a subject as a candidate for treating or inhibiting a liver disease by inhibiting the expression of a HSD17B13, by determining whether or not a sample from the subject comprises a first nucleic acid encoding a PNPLA3 protein comprising an I148M variation and a second nucleic acid encoding a functional HSD17B13 protein, and/or a PNPLA3 protein comprising an I148M variation and a functional HSD17B13 protein, and classifying the subject as a candidate for treating or inhibiting a liver disease by inhibiting HSD17B13 when both the first and second nucleic acids are detected and/or when both proteins are detected.

The variant PNPLA3 Ile148Met variant or PNPLA3 Ile144Met variant can be any of the PNPLA3 Ile148Met variants and PNPLA3 Ile144Met variants described herein. The PNPLA3 Ile148Met variant or PNPLA3 Ile144Met variant can be detected by any suitable means, such as ELISA assay, RT-PCR, sequencing.

In some embodiments, the methods further comprise determining whether the subject is homozygous or heterozygous for the PNPLA3 Ile148Met variant or the PNPLA3 Ile144Met variant. In some embodiments, the subject is homozygous for the PNPLA3 Ile148Met variant or the PNPLA3 Ile144Met variant. In some embodiments, the subject is heterozygous for the PNPLA3 Ile148Met variant or the PNPLA3 Ile144Met variant. In some embodiments, the subject is homozygous for the PNPLA3 Ile148Met variant. In some embodiments, the subject is heterozygous for the PNPLA3 Ile148Met variant. In some embodiments, the subject is homozygous for the PNPLA3 Ile144Met variant. In some embodiments, the subject is heterozygous for the PNPLA3 Ile144Met variant.

In some embodiments, the subject does not comprise any genes encoding loss of function variations in the HSD17B13 protein. It is believed that loss of function variations in the HSD17B13 protein, including those described herein and in U.S. Provisional Application Ser. No. 62/570,985, filed on Oct. 11, 2017, confer a liver disease-protective effect and it is further believed that this protective effect is enhanced in the presence of the variant PNPLA3 Ile148M variation.

In some embodiments, the methods further comprise determining whether the subject is obese. In some embodiments, a subject is obese if their body mass index (BMI) is over 30 kg/m². Obesity can be a characteristic of a subject having, or at risk of developing, a liver disease. In some embodiments, the methods further comprise determining whether the subject has a fatty liver. A fatty liver can be a characteristic of a subject having, or at risk of developing, a liver disease. In some embodiments, the methods further comprise determining whether the subject is obese and has a fatty liver.

As used herein, “nonalcoholic fatty liver disease,” used interchangeably with the term “NAFLD,” refers to a disease defined by the presence of macrovascular steatosis in the presence of less than 20 gm of alcohol ingestion per day. NAFLD is the most common liver disease in the United States, and is commonly associated with insulin resistance/type 2 diabetes mellitus and obesity. NAFLD is manifested by steatosis, steatohepatitis, cirrhosis, and sometimes hepatocellular carcinoma. For a review of NAFLD, see Tolman and Dalpiaz (2007) Ther. Clin. Risk. Manag., 3(6):1153-1163 the entire contents of which are incorporated herein by reference.

As used herein, the terms “steatosis,” “hepatic steatosis,” and “fatty liver disease” refer to the accumulation of triglycerides and other fats in the liver cells.

As used herein, the term “Nonalcoholic steatohepatitis” or “NASH” refers to liver inflammation and damage caused by a buildup of fat in the liver. NASH is part of a group of conditions called nonalcoholic fatty liver disease (NAFLD). NASH resembles alcoholic liver disease, but occurs in people who drink little or no alcohol. The major feature in NASH is fat in the liver, along with inflammation and damage. Most people with NASH feel well and are not aware that they have a liver problem. Nevertheless, NASH can be severe and can lead to cirrhosis, in which the liver is permanently damaged and scarred and no longer able to work properly. NASH is usually first suspected in a person who is found to have elevations in liver tests that are included in routine blood test panels, such as alanine aminotransferase (ALT) or aspartate aminotransferase (AST). When further evaluation shows no apparent reason for liver disease (such as medications, viral hepatitis, or excessive use of alcohol) and when x rays or imaging studies of the liver show fat, NASH is suspected. The only means of proving a diagnosis of NASH and separating it from simple fatty liver is a liver biopsy.

As used herein, the term “cirrhosis,” defined histologically, is a diffuse hepatic process characterized by fibrosis and conversion of the normal liver architecture into structurally abnormal nodules.

As used herein, the term “serum lipid” refers to any major lipid present in the blood. Serum lipids may be present in the blood either in free form or as a part of a protein complex, e.g., a lipoprotein complex. Non-limiting examples of serum lipids may include triglycerides (TG), cholesterol, such as total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low density lipoprotein cholesterol (VLDL-C) and intermediate-density lipoprotein cholesterol (IDL-C).

In one embodiment, a subject that would benefit from the reduction of the expression of HSD17B13 (and, in some embodiments, PNPLA3) is, for example, a subject that has type 2 diabetes and prediabetes, or obesity; a subject that has high levels of fats in the blood, such as cholesterol, or has high blood pressure; a subject that has certain metabolic disorders, including metabolic syndrome; a subject that has rapid weight loss; a subject that has certain infections, such as hepatitis C infection, or a subject that has been exposed to some toxins. In one embodiment, a subject that would benefit from the reduction of the expression of HSD17B13 (and, in some embodiments, PNPLA3) is, for example, a subject that is middle-aged or older; a subject that is Hispanic, non-Hispanic whites, or African Americans; a subject that takes certain drugs, such as corticosteroids and cancer drugs.

In the methods (and uses) of the invention which comprise administering to a subject a first dsRNA agent targeting HSD17B13 and a second dsRNA agent targeting PNPLA3, the first and second dsRNA agents may be formulated in the same composition or different compositions and may administered to the subject in the same composition or in separate compositions.

In one embodiment, an “iRNA” for use in the methods of the invention is a “dual targeting RNAi agent.” The term “dual targeting RNAi agent” refers to a molecule comprising a first dsRNA agent comprising a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a first target RNA, i.e., an HSD17B13 gene, covalently attached to a molecule comprising a second dsRNA agent comprising a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a second target RNA, i.e., a PNPLA3 gene. In some embodiments of the invention, a dual targeting RNAi agent triggers the degradation of the first and the second target RNAs, e.g., mRNAs, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

The dsRNA agent may be administered to the subject at a dose of about 0.1 mg/kg to about 50 mg/kg. Typically, a suitable dose will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg.

The iRNA can be administered by intravenous infusion over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis.

Administration of the iRNA can reduce HSD17B13 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or at least about 99% or more. In a preferred embodiment, administration of the iRNA can reduce HSD17B13 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 20%.

Administration of the iRNA can reduce PNPLA3 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or at least about 99% or more. In a preferred embodiment, administration of the iRNA can reduce PNPLA3 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 20%.

Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Alternatively, the iRNA can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of iRNA to a subject. The injections may be repeated over a period of time. The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day or to once a year. In certain embodiments, the iRNA is administered about once per week, once every 7-10 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once per month, once every 2 months, once every 3 months once per quarter), once every 4 months, once every 5 months, or once every 6 months.

In one embodiment, the method includes administering a composition featured herein such that expression of the target HSD17B13 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, expression of the target HSD17B13 gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer.

In another embodiment, the method includes administering a composition featured herein such that expression of the target PNPLA3 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, expression of the target PNPLA3 gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer.

Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target HSD17B13 gene (and, in some embodiments, a PNPLA3 gene). Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.

Administration of the dsRNA according to the methods of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a disorder of lipid metabolism. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a disorder of lipid metabolism may be assessed, for example, by periodic monitoring of one or more serum lipid levels, e.g., triglyceride levels. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA or pharmaceutical composition thereof, “effective against” a disorder of lipid metabolism indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating disorder of lipid metabolisms and the related causes.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art.

The invention further provides methods for the use of a iRNA agent or a pharmaceutical composition of the invention, e.g., for treating a subject that would benefit from reduction and/or inhibition of HSD17B13 expression or HSD17B13, e.g., a subject having an HSD17B13-associated disease disorder, or condition, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. In some embodiments, the invention provides methods for the use of a iRNA agent or a pharmaceutical composition of the invention and an iRNA agent targeting PNPLA3, e.g., for treating a subject that would benefit from reduction and/or inhibition of HSD17B13 expression and PNPLA3 expression, e.g., a subject having an HSD17B13-associated disease disorder, or condition (e.g., NASH), in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an iRNA agent or pharmaceutical composition of the invention is administered in combination with, e.g., pyridoxine, an ACE inhibitor (angiotensin converting enzyme inhibitors), e.g., benazepril agents to decrease blood pressure, e.g., diuretics, beta-blockers, ACE inhibitors, angiotensin II receptor blockers, calcium channel blockers, alpha blockers, alpha-2 receptor antagonists, combined alpha- and beta-blockers, central agonists, peripheral adrenergic inhibitors, and blood vessel dilators; or agents to decrease cholesterol, e.g., statins, selective cholesterol absorption inhibitors, resins; lipid lowering therapies; insulin sensitizers, such as the PPARγ agonist pioglitazone; glp-1r agonists, such as liraglutatide; vitamin E; SGLT2 inhibitors; or DPPIV inhibitors; or a combination of any of the foregoing. In one embodiment, an iRNA agent or pharmaceutical composition of the invention is administered in combination with an agent that inhibits the expression and/or activity of a transmembrane 6 superfamily member 2 (TM6SF2) gene, e.g., an RNAi agent that inhibits the expression of a TM6SF2 gene.

The iRNA agent and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., subcutaneously, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

VII. Kits

The present invention also provides kits for performing any of the methods of the invention. Such kits include one or more RNAi agent(s) and instructions for use, e.g., instructions for inhibiting expression of a HSD17B13 in a cell by contacting the cell with an RNAi agent or pharmaceutical composition of the invention in an amount effective to inhibit expression of the HSD17B13. The kits may optionally further comprise means for contacting the cell with the RNAi agent (e.g., an injection device), or means for measuring the inhibition of HSD17B13 (e.g., means for measuring the inhibition of HSD17B13 mRNA and/or HSD17B13 protein). Such means for measuring the inhibition of HSD17B13 may comprise a means for obtaining a sample from a subject, such as, e.g., a plasma sample. The kits of the invention may optionally further comprise means for administering the RNAi agent(s) to a subject or means for determining the therapeutically effective or prophylactically effective amount.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES
Example 1. iRNA Design, Synthesis, and Selection

This Example describes methods for the design, synthesis, and selection of HSD17B13 iRNA agents.

Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Bioinformatics

A set of siRNAs targeting the human hydroxysteroid 17-beta dehydrogenase 13 gene (HSD17B13; human NCBI refseqID NM_178135.4; NCBI GeneID: 345275) as well the toxicology-species HSD17B13 ortholog from cynomolgus monkey: XM_005555367.2 was designed using custom R and Python scripts. All the siRNA designs have a perfect match to the human HSD17B13 transcript and a subset either perfect or near-perfect matches to the cynomolgus monkey ortholog. The human NM_178135 REFSEQ mRNA, version 4, has a length of 2397 bases. The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 23mer siRNA from position 10 through the end was determined with a random forest model derived from the direct measure of mRNA knockdown from several thousand distinct siRNA designs targeting a diverse set of vertebrate genes. For each strand of the siRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the siRNA and all potential alignments in the human transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the siRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8, 1.2, 1 for seed mismatches, cleavage site, and other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to siRNAs whose antisense score in human and cynomolgus monkey was >=2 and predicted efficacy was >=50% knockdown.

A detailed list of the unmodified nucleotide sequences of the sense strand and antisense strand sequences is shown in Table 2.

A detailed list of the modified nucleotide sequences of the sense strand and antisense strand sequences is shown in Table 3.

In Vitro Cos-7 (Dual-Luciferase psiCHECK2 Vector), Primary Human Hepatocytes, and Primary Cynomolgus Hepatocytes Screening

Cell Culture and Transfections:

Cos-7 (ATCC) were transfected by adding 5 μl of 1 ng/ul, diluted in Opti-MEM, HSD17B13 psiCHECK2 vector (Blue Heron Biotechnology), 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine 2000 per well (Invitrogen, Carlsbad Calif. cat #11668-019) to 5 μl of siRNA duplexes per well, with 4 replicates of each siRNA duplex, into a 384-well plate, and incubated at room temperature for 15 minutes. Thirty-five μl of Dulbecco's Modified Eagle Medium (ThermoFisher) containing ˜5×10³cells were then added to the siRNA mixture. Cells were incubated for 48 hours followed by Firefly (transfection control) and Renilla (fused to target sequence) luciferase measurements. Single dose experiments were performed at 50 nM.

Primary Human Hepatocytes (BioIVT) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of RNAiMAX per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well, with 4 replicates of each siRNA duplex, into a 384-well plate, and incubated at room temperature for 15 minutes. Firthy μl of InVitroGRO CP plating media (BioIVT) containing ˜15×10³cells were then added to the siRNA mixture. Cells were incubated for 48 hours prior to RNA purification. Single dose experiments were performed at 50 nM.

Primary Cynomolgus Hepatocytes (BioIVT) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of RNAiMAX per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well, with 4 replicates of each siRNA duplex, into a 384-well plate, and incubated at room temperature for 15 minutes. Firthy μl of InVitroGRO CP plating media (BioIVT) containing ˜5×10³cells were then added to the siRNA mixture. Cells were incubated for 48 hours prior to RNA purification. Single dose experiments were performed at 50 nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit:

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 70 ul of Lysis/Binding Buffer and 10 ul of lysis buffer containing 3 ul of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150 ul Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 ul Elution Buffer, re-captured and supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813):

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 ul 25×dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H2O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 h 37° C.

Real Time PCR:

Two μl of cDNA and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) were added to either 0.5 μl of Human GAPDH TaqMan Probe (4326317E) and 0.5 μl HSD17B13 Human probe (Hs01068199_m1, Thermo) or 0.5 μl Cyno GAPDH (custom) and 0.5 μl HSD17B13 Cyno probe (Mf02888851_m1, Thermo) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). Each duplex was tested at least two times and data were normalized to cells transfected with a non-targeting control siRNA. To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with a non-targeting control siRNA.

Table 4 shows the results of a 50 nM single dose screen in Cos-7 (Dual-Luciferase psiCHECK2 vector) cells transfected with the indicate iRNA agents. Data are expressed as percent of message remaining relative to untreated cells.

Table 5 shows the results of a 50 nM single dose screen in Primary Human Hepatocytes transfected with the indicate iRNA agents. Data are expressed as percent of message remaining relative to untreated cells.

Table 6 shows the results of a 50 nM single dose screen in Primary Cynomolgus Hepatocytes transfected with the indicate iRNA agents. Data are expressed as percent of message remaining relative to untreated cells.

TABLE 1

Abbreviations of nucleotide monomers used in nucleic acid sequence representation.

It will be understood that these monomers, when present in an oligonucleotide, are mutually

linked by 5′-3′-phosphodiester bonds.

Abbreviation
Nucleotide(s)

A
Adenosine-3′-phosphate

Ab
beta-L-adenosine-3′-phosphate

Abs
beta-L-adenosine-3′-phosphorothioate

Af
2′-fluoroadenosine-3′-phosphate

Afs
2′-fluoroadenosine-3′-phosphorothioate

As
adenosine-3′-phosphorothioate

C
cytidine-3′-phosphate

Cb
beta-L-cytidine-3′-phosphate

Cbs
beta-L-cytidine-3′-phosphorothioate

Cf
2′-fluorocytidine-3′-phosphate

Cfs
2′-fluorocytidine-3′-phosphorothioate

Cs
cytidine-3′-phosphorothioate

G
guanosine-3′-phosphate

Gb
beta-L-guanosine-3′-phosphate

Gbs
beta-L-guanosine-3′-phosphorothioate

Gf
2′-fluoroguanosine-3′-phosphate

Gfs
2′-fluoroguanosine-3′-phosphorothioate

Gs
guanosine-3′-phosphorothioate

T
5′-methyluridine-3′-phosphate

Tf
2′-fluoro-5-methyluridine-3′-phosphate

Tfs
2′-fluoro-5-methyluridine-3′-phosphorothioate

Ts
5-methyluridine-3′-phosphorothioate

U
Uridine-3′-phosphate

Uf
2′-fluorouridine-3′-phosphate

Ufs
2′-fluorouridine-3′-phosphorothioate

Us
uridine-3′-phosphorothioate

N
any nucleotide (G, A, C, T or U)

a
2′-O-methyladenosine-3′-phosphate

as
2′-O-methyladenosine-3′-phosphorothioate

c
2′-O-methylcytidine-3′-phosphate

cs
2′-O-methylcytidine-3′-phosphorothioate

g
2′-O-methylguanosine-3′-phosphate

gs
2′-O-methylguanosine-3′-phosphorothioate

t
2′-O-methyl-5-methyluridine-3′-phosphate

ts
2′-O-methyl-5-methyluridine-3′-phosphorothioate

u
2′-O-methyluridine-3′-phosphate

us
2′-O-methyluridine-3′-phosphorothioate

s
phosphorothioate linkage

L96
N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol

P
Phosphate

VP
Vinyl-phosphate

dA
2′-deoxyadenosine-3-phosphate

dAs
2′-deoxyadenosine-3′-phosphorothioate

dC
2-deoxycytidine-3′-phosphate

dCs
2′-deoxycytidine-3′-phosphorothioate

dG
2′-deoxyguanosine-3′-phosphate

dGs
2′-deoxyguanosine-3′-phosphorothioate

dT
2′-deoxythymidine-3′-phosphate

dTs
2′-deoxythymidine-3′-phosphorothioate

dU
2′-deoxyuridine

dUs
2′-deoxyuridine-3′-phosphorothioate

Y34
2-hydroxymethyl-tetrahydrofurane-4-methoxy-3-phosphate (abasic 2′-

OMe furanose)

Y44
inverted abasic DNA (2-hydroxymethyl-tetrahydrofurane-5-phosphate)

(Agn)
Adenosine-glycol nucleic acid (GNA)

(Cgn)
Cytidine-glycol nucleic acid (GNA)

(Ggn)
Guanosine-glycol nucleic acid (GNA)

(Tgn)
Thymidine-glycol nucleic acid (GNA) S-Isomer

(Aam)
2′-O-(N-methylacetamide)adenosine-3′-phosphate

(Aams)
2′-O-(N-methylacetamide)adenosine-3′-phosphorothioate

(Gam)
2′-O-(N-methylacetamide)guanosine-3′-phosphate

(Gams)
2′-O-(N-methylacetamide)guanosine-3′-phosphorothioate

(Tam)
2′-O-(N-methylacetamide)thymidine-3′-phosphate

(Tams)
2′-O-(N-methylacetamide)thymidine-3′-phosphorothioate

(Aeo)
2′-O-methoxyethyladenosine-3′-phosphate

(Aeos)
2′-O-methoxyethyladenosine-3′-phosphorothioate

(Geo)
2′-O-methoxyethylguanosine-3′-phosphate

(Geos)
2′-O-methoxyethylguanosine-3′-phosphorothioate

(Teo)
2′-O-methoxyethyl-5-methyluridine-3′-phosphate

(Teos)
2′-O-methoxyethyl-5-methyluridine-3′-phosphorothioate

(m5Ceo)
2′-O-methoxyethyl-5-methylcytidine-3′-phosphate

(m5Ceos)
2′-O-methoxyethyl-5-methylcytidine-3′-phosphorothioate

(A3m)
3′-O-methyladenosine-2′-phosphate

(A3mx)
3′-O-methyl-xylofuranosyladenosine-2′-phosphate

(G3m)
3′-O-methylguanosine-2′-phosphate

(G3mx)
3′-O-methyl-xylofuranosylguanosine-2′-phosphate

(C3m)
3′-O-methylcytidine-2′-phosphate

(C3mx)
3′-O-methyl-xylofuranosylcytidine-2′-phosphate

(U3m)
3′-O-methyluridine-2′-phosphate

U3mx)
3′-O-methyl-xylofuranosyluridine-2′-phosphate

(m5Cam)
2′-O-(N-methylacetamide)-5-methylcytidine-3′-phosphate

(m5Cams)
2′-O-(N-methylacetamide)-5-methylcytidine-3′-phosphorothioate

(Chd)
2′-O-hexadecyl-cytidine-3′-phosphate

(Chds)
2′-O-hexadecyl-cytidine-3′-phosphorothioate

(Uhd)
2′-O-hexadecyl-uridine-3′-phosphate

(Uhds)
2′-O-hexadecyl-uridine-3′-phosphorothioate

(pshe)
Hydroxyethylphosphorothioate

¹The chemical structure of L96 is as follows:

embedded image

TABLE 2

HSD17B13 Unmodified Sequences

SEQ

SEQ

ID
Sense Strand Sequence
Range in
ID
Antisense Strand Sequence
Range in

Duplex Name
NO:
5′ to 3′
NM_178135.4
NO:
5′ to 3′
NM_178135.4

AD-238911.1
3003
CAGUACCUCCUCCCUAGGACU
4-24
3299
AGUCCUAGGGAGGAGGUACUGUC
2-24

AD-238912.1
3004
AGUACCUCCUCCCUAGGACUA
5-25
3300
UAGUCCUAGGGAGGAGGUACUGU
3-25

AD-238913.1
3005
GUACCUCCUCCCUAGGACUAA
6-26
3301
UUAGUCCUAGGGAGGAGGUACUG
4-26

AD-238929.1
3006
ACUACACAAGGACUGAACCAA
22-42
3302
UUGGUUCAGUCCUUGUGUAGUCC
20-42

AD-238930.1
3007
CUACACAAGGACUGAACCAGA
23-43
3303
UCUGGUUCAGUCCUUGUGUAGUC
21-43

AD-238932.1
3008
ACACAAGGACUGAACCAGAAA
25-45
3304
UUUCUGGUUCAGUCCUUGUGUAG
23-45

AD-238960.1
3009
CAGAGCAAAGCCAUGAACAUA
54-74
3305
UAUGUUCAUGGCUUUGCUCUGUC
52-74

AD-238979.1
3010
UCAUCCUAGAAAUCCUUCUGA
73-93
3306
UCAGAAGGAUUUCUAGGAUGAUG
71-93

AD-238991.1
3011
UCCUUCUGCUUCUGAUCACCA
85-105
3307
UGGUGAUCAGAAGCAGAAGGAUU
83-105

AD-239009.1
3012
CCAUCAUCUACUCCUACUUGA
103-123
3308
UCAAGUAGGAGUAGAUGAUGGUG
101-123

AD-239015.1
3013
UCUACUCCUACUUGGAGUCGU
109-129
3309
ACGACUCCAAGUAGGAGUAGAUG
107-129

AD-239016.1
3014
CUACUCCUACUUGGAGUCGUU
110-130
3310
AACGACUCCAAGUAGGAGUAGAU
108-130

AD-239017.1
3015
UACUCCUACUUGGAGUCGUUA
111-131
3311
UAACGACUCCAAGUAGGAGUAGA
109-131

AD-239018.1
3016
ACUCCUACUUGGAGUCGUUGA
112-132
3312
UCAACGACUCCAAGUAGGAGUAG
110-132

AD-239019.1
3017
CUCCUACUUGGAGUCGUUGGU
113-133
3313
ACCAACGACUCCAAGUAGGAGUA
111-133

AD-239020.1
3018
UCCUACUUGGAGUCGUUGGUA
114-134
3314
UACCAACGACUCCAAGUAGGAGU
112-134

AD-239031.1
3019
GUCGUUGGUGAAGUUUUUCAU
125-145
3315
AUGAAAAACUUCACCAACGACUC
123-145

AD-239062.1
3020
GAGAUUGUUCUCAUUACUGGA
174-194
3316
UCCAGUAAUGAGAACAAUCUCCC
172-194

AD-239066.1
3021
UUGUUCUCAUUACUGGAGCUA
178-198
3317
UAGCUCCAGUAAUGAGAACAAUC
176-198

AD-239103.1
3022
GCAGACUACUUAUGAAUUUGA
215-235
3318
UCAAAUUCAUAAGUAGUCUGCCU
213-235

AD-239110.1
3023
ACUUAUGAAUUUGCAAAACGA
222-242
3319
UCGUUUUGCAAAUUCAUAAGUAG
220-242

AD-239118.1
3024
AUUUGCAAAACGACAGAGCAU
230-250
3320
AUGCUCUGUCGUUUUGCAAAUUC
228-250

AD-239119.1
3025
UUUGCAAAACGACAGAGCAUA
231-251
3321
UAUGCUCUGUCGUUUUGCAAAUU
229-251

AD-239131.1
3026
CAGAGCAUAUUGGUUCUGUGA
243-263
3322
UCACAGAACCAAUAUGCUCUGUC
241-263

AD-239132.1
3027
AGAGCAUAUUGGUUCUGUGGA
244-264
3323
UCCACAGAACCAAUAUGCUCUGU
242-264

AD-239133.1
3028
GAGCAUAUUGGUUCUGUGGGA
245-265
3324
UCCCACAGAACCAAUAUGCUCUG
243-265

AD-239146.1
3029
CUGUGGGAUAUUAAUAAGCGA
258-278
3325
UCGCUUAUUAAUAUCCCACAGAA
256-278

AD-239155.1
3030
AUUAAUAAGCGCGGUGUGGAA
267-287
3326
UUCCACACCGCGCUUAUUAAUAU
265-287

AD-239156.1
3031
UUAAUAAGCGCGGUGUGGAGA
268-288
3327
UCUCCACACCGCGCUUAUUAAUA
266-288

AD-239177.1
3032
AAACUGCAGCUGAGUGCCGAA
289-309
3328
UUCGGCACUCAGCUGCAGUUUCC
287-309

AD-239178.1
3033
AACUGCAGCUGAGUGCCGAAA
290-310
3329
UUUCGGCACUCAGCUGCAGUUUC
288-310

AD-239179.1
3034
ACUGCAGCUGAGUGCCGAAAA
291-311
3330
UUUUCGGCACUCAGCUGCAGUUU
289-311

AD-239180.1
3035
CUGCAGCUGAGUGCCGAAAAC
292-312
3331
GUUUUCGGCACUCAGCUGCAGUU
290-312

AD-239181.1
3036
UGCAGCUGAGUGCCGAAAACU
293-313
3332
AGUUUUCGGCACUCAGCUGCAGU
291-313

AD-239182.1
3037
GCAGCUGAGUGCCGAAAACUA
294-314
3333
UAGUUUUCGGCACUCAGCUGCAG
292-314

AD-239183.1
3038
CAGCUGAGUGCCGAAAACUAA
295-315
3334
UUAGUUUUCGGCACUCAGCUGCA
293-315

AD-239185.1
3039
GCUGAGUGCCGAAAACUAGGA
297-317
3335
UCCUAGUUUUCGGCACUCAGCUG
295-317

AD-239186.1
3040
CUGAGUGCCGAAAACUAGGCA
298-318
3336
UGCCUAGUUUUCGGCACUCAGCU
296-318

AD-239187.1
3041
UGAGUGCCGAAAACUAGGCGU
299-319
3337
ACGCCUAGUUUUCGGCACUCAGC
297-319

AD-239188.1
3042
GAGUGCCGAAAACUAGGCGUA
300-320
3338
UACGCCUAGUUUUCGGCACUCAG
298-320

AD-239189.1
3043
AGUGCCGAAAACUAGGCGUCA
301-321
3339
UGACGCCUAGUUUUCGGCACUCA
299-321

AD-239190.1
3044
GUGCCGAAAACUAGGCGUCAA
302-322
3340
UUGACGCCUAGUUUUCGGCACUC
300-322

AD-239191.1
3045
UGCCGAAAACUAGGCGUCACU
303-323
3341
AGUGACGCCUAGUUUUCGGCACU
301-323

AD-239192.1
3046
GCCGAAAACUAGGCGUCACUA
304-324
3342
UAGUGACGCCUAGUUUUCGGCAC
302-324

AD-239193.1
3047
CCGAAAACUAGGCGUCACUGA
305-325
3343
UCAGUGACGCCUAGUUUUCGGCA
303-325

AD-239194.1
3048
CGAAAACUAGGCGUCACUGCA
306-326
3344
UGCAGUGACGCCUAGUUUUCGGC
304-326

AD-239196.1
3049
AAAACUAGGCGUCACUGCGCA
308-328
3345
UGCGCAGUGACGCCUAGUUUUCG
306-328

AD-239198.1
3050
AACUAGGCGUCACUGCGCAUA
310-330
3346
UAUGCGCAGUGACGCCUAGUUUU
308-330

AD-239199.1
3051
ACUAGGCGUCACUGCGCAUGA
311-331
3347
UCAUGCGCAGUGACGCCUAGUUU
309-331

AD-239203.1
3052
GGCGUCACUGCGCAUGCGUAU
315-335
3348
AUACGCAUGCGCAGUGACGCCUA
313-335

AD-239214.1
3053
GCAUGCGUAUGUGGUAGACUA
326-346
3349
UAGUCUACCACAUACGCAUGCGC
324-346

AD-239215.1
3054
CAUGCGUAUGUGGUAGACUGA
327-347
3350
UCAGUCUACCACAUACGCAUGCG
325-347

AD-239239.1
3055
AACAGAGAAGAGAUCUAUCGA
351-371
3351
UCGAUAGAUCUCUUCUCUGUUGC
349-371

AD-239240.1
3056
ACAGAGAAGAGAUCUAUCGCU
352-372
3352
AGCGAUAGAUCUCUUCUCUGUUG
350-372

AD-239241.1
3057
CAGAGAAGAGAUCUAUCGCUA
353-373
3353
UAGCGAUAGAUCUCUUCUCUGUU
351-373

AD-239242.1
3058
AGAGAAGAGAUCUAUCGCUCU
354-374
3354
AGAGCGAUAGAUCUCUUCUCUGU
352-374

AD-239243.1
3059
GAGAAGAGAUCUAUCGCUCUA
355-375
3355
UAGAGCGAUAGAUCUCUUCUCUG
353-375

AD-239245.1
3060
GAAGAGAUCUAUCGCUCUCUA
357-377
3356
UAGAGAGCGAUAGAUCUCUUCUC
355-377

AD-239246.1
3061
AAGAGAUCUAUCGCUCUCUAA
358-378
3357
UUAGAGAGCGAUAGAUCUCUUCU
356-378

AD-239247.1
3062
AGAGAUCUAUCGCUCUCUAAA
359-379
3358
UUUAGAGAGCGAUAGAUCUCUUC
357-379

AD-239250.1
3063
GAUCUAUCGCUCUCUAAAUCA
362-382
3359
UGAUUUAGAGAGCGAUAGAUCUC
360-382

AD-239251.1
3064
AUCUAUCGCUCUCUAAAUCAA
363-383
3360
UUGAUUUAGAGAGCGAUAGAUCU
361-383

AD-239253.1
3065
CUAUCGCUCUCUAAAUCAGGU
365-385
3361
ACCUGAUUUAGAGAGCGAUAGAU
363-385

AD-239254.1
3066
UAUCGCUCUCUAAAUCAGGUA
366-386
3362
UACCUGAUUUAGAGAGCGAUAGA
364-386

AD-239255.1
3067
AUCGCUCUCUAAAUCAGGUGA
367-387
3363
UCACCUGAUUUAGAGAGCGAUAG
365-387

AD-239290.1
3068
GAUGUAACAAUCGUGGUGAAU
402-422
3364
AUUCACCACGAUUGUUACAUCAC
400-422

AD-239291.1
3069
AUGUAACAAUCGUGGUGAAUA
403-423
3365
UAUUCACCACGAUUGUUACAUCA
401-423

AD-239292.1
3070
UGUAACAAUCGUGGUGAAUAA
404-424
3366
UUAUUCACCACGAUUGUUACAUC
402-424

AD-239293.1
3071
GUAACAAUCGUGGUGAAUAAU
405-425
3367
AUUAUUCACCACGAUUGUUACAU
403-425

AD-239304.1
3072
GGUGAAUAAUGCUGGGACAGU
416-436
3368
ACUGUCCCAGCAUUAUUCACCAC
414-436

AD-239312.1
3073
AUGCUGGGACAGUAUAUCCAA
424-444
3369
UUGGAUAUACUGUCCCAGCAUUA
422-444

AD-239313.1
3074
UGCUGGGACAGUAUAUCCAGA
425-445
3370
UCUGGAUAUACUGUCCCAGCAUU
423-445

AD-239314.1
3075
GCUGGGACAGUAUAUCCAGCA
426-446
3371
UGCUGGAUAUACUGUCCCAGCAU
424-446

AD-239316.1
3076
UGGGACAGUAUAUCCAGCCGA
428-448
3372
UCGGCUGGAUAUACUGUCCCAGC
426-448

AD-239317.1
3077
GGGACAGUAUAUCCAGCCGAU
429-449
3373
AUCGGCUGGAUAUACUGUCCCAG
427-449

AD-239318.1
3078
GGACAGUAUAUCCAGCCGAUA
430-450
3374
UAUCGGCUGGAUAUACUGUCCCA
428-450

AD-239321.1
3079
CAGUAUAUCCAGCCGAUCUUA
433-453
3375
UAAGAUCGGCUGGAUAUACUGUC
431-453

AD-239332.1
3080
GCCGAUCUUCUCAGCACCAAA
444-464
3376
UUUGGUGCUGAGAAGAUCGGCUG
442-464

AD-239337.1
3081
UCUUCUCAGCACCAAGGAUGA
449-469
3377
UCAUCCUUGGUGCUGAGAAGAUC
447-469

AD-239348.1
3082
CCAAGGAUGAAGAGAUUACCA
460-480
3378
UGGUAAUCUCUUCAUCCUUGGUG
458-480

AD-239362.1
3083
AUUACCAAGACAUUUGAGGUA
474-494
3379
UACCUCAAAUGUCUUGGUAAUCU
472-494

AD-239366.1
3084
CCAAGACAUUUGAGGUCAACA
478-498
3380
UGUUGACCUCAAAUGUCUUGGUA
476-498

AD-239368.1
3085
AAGACAUUUGAGGUCAACAUA
480-500
3381
UAUGUUGACCUCAAAUGUCUUGG
478-500

AD-239372.1
3086
CAUUUGAGGUCAACAUCCUAA
484-504
3382
UUAGGAUGUUGACCUCAAAUGUC
482-504

AD-239373.1
3087
AUUUGAGGUCAACAUCCUAGA
485-505
3383
UCUAGGAUGUUGACCUCAAAUGU
483-505

AD-239379.1
3088
GGUCAACAUCCUAGGACAUUU
491-511
3384
AAAUGUCCUAGGAUGUUGACCUC
489-511

AD-239380.1
3089
GUCAACAUCCUAGGACAUUUU
492-512
3385
AAAAUGUCCUAGGAUGUUGACCU
490-512

AD-239387.1
3090
UCCUAGGACAUUUUUGGAUCA
499-519
3386
UGAUCCAAAAAUGUCCUAGGAUG
497-519

AD-239397.1
3091
UUUUUGGAUCACAAAAGCACU
509-529
3387
AGUGCUUUUGUGAUCCAAAAAUG
507-529

AD-239411.1
3092
AAGCACUUCUUCCAUCGAUGA
523-543
3388
UCAUCGAUGGAAGAAGUGCUUUU
521-543

AD-239412.1
3093
AGCACUUCUUCCAUCGAUGAU
524-544
3389
AUCAUCGAUGGAAGAAGUGCUUU
522-544

AD-239431.1
3094
AUGGAGAGAAAUCAUGGCCAA
543-563
3390
UUGGCCAUGAUUUCUCUCCAUCA
541-563

AD-239433.1
3095
GGAGAGAAAUCAUGGCCACAU
545-565
3391
AUGUGGCCAUGAUUUCUCUCCAU
543-565

AD-239436.1
3096
GAGAAAUCAUGGCCACAUCGU
548-568
3392
ACGAUGUGGCCAUGAUUUCUCUC
546-568

AD-239437.1
3097
AGAAAUCAUGGCCACAUCGUA
549-569
3393
UACGAUGUGGCCAUGAUUUCUCU
547-569

AD-239438.1
3098
GAAAUCAUGGCCACAUCGUCA
550-570
3394
UGACGAUGUGGCCAUGAUUUCUC
548-570

AD-239439.1
3099
AAAUCAUGGCCACAUCGUCAA
551-571
3395
UUGACGAUGUGGCCAUGAUUUCU
549-571

AD-239450.1
3100
ACAUCGUCACAGUGGCUUCAA
562-582
3396
UUGAAGCCACUGUGACGAUGUGG
560-582

AD-239451.1
3101
CAUCGUCACAGUGGCUUCAGU
563-583
3397
ACUGAAGCCACUGUGACGAUGUG
561-583

AD-239452.1
3102
AUCGUCACAGUGGCUUCAGUA
564-584
3398
UACUGAAGCCACUGUGACGAUGU
562-584

AD-239453.1
3103
UCGUCACAGUGGCUUCAGUGU
565-585
3399
ACACUGAAGCCACUGUGACGAUG
563-585

AD-239475.1
3104
CGGCCACGAAGGGAUUCCUUA
587-607
3400
UAAGGAAUCCCUUCGUGGCCGCA
585-607

AD-239476.1
3105
GGCCACGAAGGGAUUCCUUAA
588-608
3401
UUAAGGAAUCCCUUCGUGGCCGC
586-608

AD-239478.1
3106
CCACGAAGGGAUUCCUUACCU
590-610
3402
AGGUAAGGAAUCCCUUCGUGGCC
588-610

AD-239479.1
3107
CACGAAGGGAUUCCUUACCUA
591-611
3403
UAGGUAAGGAAUCCCUUCGUGGC
589-611

AD-239480.1
3108
ACGAAGGGAUUCCUUACCUCA
592-612
3404
UGAGGUAAGGAAUCCCUUCGUGG
590-612

AD-239482.1
3109
GAAGGGAUUCCUUACCUCAUA
594-614
3405
UAUGAGGUAAGGAAUCCCUUCGU
592-614

AD-239483.1
3110
AAGGGAUUCCUUACCUCAUCA
595-615
3406
UGAUGAGGUAAGGAAUCCCUUCG
593-615

AD-239499.1
3111
CAUCCCAUAUUGUUCCAGCAA
611-631
3407
UUGCUGGAACAAUAUGGGAUGAG
609-631

AD-239510.1
3112
GUUCCAGCAAAUUUGCCGCUA
622-642
3408
UAGCGGCAAAUUUGCUGGAACAA
620-642

AD-239520.1
3113
AUUUGCCGCUGUUGGCUUUCA
632-652
3409
UGAAAGCCAACAGCGGCAAAUUU
630-652

AD-239521.1
3114
UUUGCCGCUGUUGGCUUUCAA
633-653
3410
UUGAAAGCCAACAGCGGCAAAUU
631-653

AD-239522.1
3115
UUGCCGCUGUUGGCUUUCACA
634-654
3411
UGUGAAAGCCAACAGCGGCAAAU
632-654

AD-239534.1
3116
GCUUUCACAGAGGUCUGACAU
646-666
3412
AUGUCAGACCUCUGUGAAAGCCA
644-666

AD-239539.1
3117
CACAGAGGUCUGACAUCAGAA
651-671
3413
UUCUGAUGUCAGACCUCUGUGAA
649-671

AD-239544.1
3118
AGGUCUGACAUCAGAACUUCA
656-676
3414
UGAAGUUCUGAUGUCAGACCUCU
654-676

AD-239566.1
3119
GCCUUGGGAAAAACUGGUAUA
678-698
3415
UAUACCAGUUUUUCCCAAGGCCU
676-698

AD-239578.1
3120
ACUGGUAUCAAAACCUCAUGU
690-710
3416
ACAUGAGGUUUUGAUACCAGUUU
688-710

AD-239579.1
3121
CUGGUAUCAAAACCUCAUGUA
691-711
3417
UACAUGAGGUUUUGAUACCAGUU
689-711

AD-239611.1
3122
UUUGUGAAUACUGGGUUCACA
723-743
3418
UGUGAACCCAGUAUUCACAAAAA
721-743

AD-239612.1
3123
UUGUGAAUACUGGGUUCACCA
724-744
3419
UGGUGAACCCAGUAUUCACAAAA
722-744

AD-239613.1
3124
UGUGAAUACUGGGUUCACCAA
725-745
3420
UUGGUGAACCCAGUAUUCACAAA
723-745

AD-239617.1
3125
AAUACUGGGUUCACCAAAAAU
729-749
3421
AUUUUUGGUGAACCCAGUAUUCA
727-749

AD-239618.1
3126
AUACUGGGUUCACCAAAAAUA
730-750
3422
UAUUUUUGGUGAACCCAGUAUUC
728-750

AD-239637.1
3127
UCCAAGCACAAGAUUAUGGCA
749-769
3423
UGCCAUAAUCUUGUGCUUGGAUU
747-769

AD-239641.1
3128
AGCACAAGAUUAUGGCCUGUA
753-773
3424
UACAGGCCAUAAUCUUGUGCUUG
751-773

AD-239642.1
3129
GCACAAGAUUAUGGCCUGUAU
754-774
3425
AUACAGGCCAUAAUCUUGUGCUU
752-774

AD-239643.1
3130
CACAAGAUUAUGGCCUGUAUU
755-775
3426
AAUACAGGCCAUAAUCUUGUGCU
753-775

AD-239645.1
3131
CAAGAUUAUGGCCUGUAUUGA
757-777
3427
UCAAUACAGGCCAUAAUCUUGUG
755-777

AD-239647.1
3132
AGAUUAUGGCCUGUAUUGGAA
759-779
3428
UUCCAAUACAGGCCAUAAUCUUG
757-779

AD-239649.1
3133
AUUAUGGCCUGUAUUGGAGAA
761-781
3429
UUCUCCAAUACAGGCCAUAAUCU
759-781

AD-239650.1
3134
UUAUGGCCUGUAUUGGAGACA
762-782
3430
UGUCUCCAAUACAGGCCAUAAUC
760-782

AD-239653.1
3135
UGGCCUGUAUUGGAGACAGAU
765-785
3431
AUCUGUCUCCAAUACAGGCCAUA
763-785

AD-239654.1
3136
GGCCUGUAUUGGAGACAGAUA
766-786
3432
UAUCUGUCUCCAAUACAGGCCAU
764-786

AD-239662.1
3137
UUGGAGACAGAUGAAGUCGUA
774-794
3433
UACGACUUCAUCUGUCUCCAAUA
772-794

AD-239668.1
3138
ACAGAUGAAGUCGUAAGAAGU
780-800
3434
ACUUCUUACGACUUCAUCUGUCU
778-800

AD-239669.1
3139
CAGAUGAAGUCGUAAGAAGUA
781-801
3435
UACUUCUUACGACUUCAUCUGUC
779-801

AD-239670.1
3140
AGAUGAAGUCGUAAGAAGUCU
782-802
3436
AGACUUCUUACGACUUCAUCUGU
780-802

AD-239671.1
3141
GAUGAAGUCGUAAGAAGUCUA
783-803
3437
UAGACUUCUUACGACUUCAUCUG
781-803

AD-239674.1
3142
GAAGUCGUAAGAAGUCUGAUA
786-806
3438
UAUCAGACUUCUUACGACUUCAU
784-806

AD-239676.1
3143
AGUCGUAAGAAGUCUGAUAGA
788-808
3439
UCUAUCAGACUUCUUACGACUUC
786-808

AD-239681.1
3144
UAAGAAGUCUGAUAGAUGGAA
793-813
3440
UUCCAUCUAUCAGACUUCUUACG
791-813

AD-239682.1
3145
AAGAAGUCUGAUAGAUGGAAU
794-814
3441
AUUCCAUCUAUCAGACUUCUUAC
792-814

AD-239684.1
3146
GAAGUCUGAUAGAUGGAAUAA
796-816
3442
UUAUUCCAUCUAUCAGACUUCUU
794-816

AD-239685.1
3147
AAGUCUGAUAGAUGGAAUACU
797-817
3443
AGUAUUCCAUCUAUCAGACUUCU
795-817

AD-239697.1
3148
UGGAAUACUUACCAAUAAGAA
809-829
3444
UUCUUAUUGGUAAGUAUUCCAUC
807-829

AD-239713.1
3149
AAUGAUUUUUGUUCCAUCGUA
830-850
3445
UACGAUGGAACAAAAAUCAUUUU
828-850

AD-239714.1
3150
AUGAUUUUUGUUCCAUCGUAU
831-851
3446
AUACGAUGGAACAAAAAUCAUUU
829-851

AD-239717.1
3151
AUUUUUGUUCCAUCGUAUAUA
834-854
3447
UAUAUACGAUGGAACAAAAAUCA
832-854

AD-239721.1
3152
UUGUUCCAUCGUAUAUCAAUA
838-858
3448
UAUUGAUAUACGAUGGAACAAAA
836-858

AD-239723.1
3153
GUUCCAUCGUAUAUCAAUAUA
840-860
3449
UAUAUUGAUAUACGAUGGAACAA
838-860

AD-239724.1
3154
UUCCAUCGUAUAUCAAUAUCU
841-861
3450
AGAUAUUGAUAUACGAUGGAACA
839-861

AD-239725.1
3155
UCCAUCGUAUAUCAAUAUCUU
842-862
3451
AAGAUAUUGAUAUACGAUGGAAC
840-862

AD-239726.1
3156
CCAUCGUAUAUCAAUAUCUUU
843-863
3452
AAAGAUAUUGAUAUACGAUGGAA
841-863

AD-239755.1
3157
ACAGAAGUUUCUUCCUGAACA
872-892
3453
UGUUCAGGAAGAAACUUCUGUAG
870-892

AD-239757.1
3158
AGAAGUUUCUUCCUGAACGCA
874-894
3454
UGCGUUCAGGAAGAAACUUCUGU
872-894

AD-239759.1
3159
AAGUUUCUUCCUGAACGCGCA
876-896
3455
UGCGCGUUCAGGAAGAAACUUCU
874-896

AD-239761.1
3160
GUUUCUUCCUGAACGCGCCUA
878-898
3456
UAGGCGCGUUCAGGAAGAAACUU
876-898

AD-239764.1
3161
UCUUCCUGAACGCGCCUCAGA
881-901
3457
UCUGAGGCGCGUUCAGGAAGAAA
879-901

AD-239778.1
3162
CCUCAGCGAUUUUAAAUCGUA
895-915
3458
UACGAUUUAAAAUCGCUGAGGCG
893-915

AD-239779.1
3163
CUCAGCGAUUUUAAAUCGUAU
896-916
3459
AUACGAUUUAAAAUCGCUGAGGC
894-916

AD-239780.1
3164
UCAGCGAUUUUAAAUCGUAUA
897-917
3460
UAUACGAUUUAAAAUCGCUGAGG
895-917

AD-239781.1
3165
CAGCGAUUUUAAAUCGUAUGA
898-918
3461
UCAUACGAUUUAAAAUCGCUGAG
896-918

AD-239782.1
3166
AGCGAUUUUAAAUCGUAUGCA
899-919
3462
UGCAUACGAUUUAAAAUCGCUGA
897-919

AD-239783.1
3167
GCGAUUUUAAAUCGUAUGCAA
900-920
3463
UUGCAUACGAUUUAAAAUCGCUG
898-920

AD-239784.1
3168
CGAUUUUAAAUCGUAUGCAGA
901-921
3464
UCUGCAUACGAUUUAAAAUCGCU
899-921

AD-239785.1
3169
GAUUUUAAAUCGUAUGCAGAA
902-922
3465
UUCUGCAUACGAUUUAAAAUCGC
900-922

AD-239786.1
3170
AUUUUAAAUCGUAUGCAGAAU
903-923
3466
AUUCUGCAUACGAUUUAAAAUCG
901-923

AD-239787.1
3171
UUUUAAAUCGUAUGCAGAAUA
904-924
3467
UAUUCUGCAUACGAUUUAAAAUC
902-924

AD-239791.1
3172
AAAUCGUAUGCAGAAUAUUCA
908-928
3468
UGAAUAUUCUGCAUACGAUUUAA
906-928

AD-239793.1
3173
AUCGUAUGCAGAAUAUUCAAU
910-930
3469
AUUGAAUAUUCUGCAUACGAUUU
908-930

AD-239806.1
3174
UAUUCAAUUUGAAGCAGUGGU
923-943
3470
ACCACUGCUUCAAAUUGAAUAUU
921-943

AD-239839.1
3175
GAAUAAAUAAGCUCCAGCCAA
967-987
3471
UUGGCUGGAGCUUAUUUAUUCAU
965-987

AD-239840.1
3176
AAUAAAUAAGCUCCAGCCAGA
968-988
3472
UCUGGCUGGAGCUUAUUUAUUCA
966-988

AD-239854.1
3177
AGCCAGAGAUGUAUGCAUGAU
982-1002
3473
AUCAUGCAUACAUCUCUGGCUGG
980-1002

AD-239855.1
3178
GCCAGAGAUGUAUGCAUGAUA
983-1003
3474
UAUCAUGCAUACAUCUCUGGCUG
981-1003

AD-239876.1
3179
AUAUGAAUAGUUUCGAAUCAA
1007-1027
3475
UUGAUUCGAAACUAUUCAUAUCA
1005-1027

AD-239877.1
3180
UAUGAAUAGUUUCGAAUCAAU
1008-1028
3476
AUUGAUUCGAAACUAUUCAUAUC
1006-1028

AD-239881.1
3181
AAUAGUUUCGAAUCAAUGCUA
1012-1032
3477
UAGCAUUGAUUCGAAACUAUUCA
1010-1032

AD-239905.1
3182
UUCAGUCCUGAUAAUAUUAAA
1054-1074
3478
UUUAAUAUUAUCAGGACUGAAAA
1052-1074

AD-239914.1
3183
AAAAACAUUGGUUUGGCACUA
1072-1092
3479
UAGUGCCAAACCAAUGUUUUUAA
1070-1092

AD-239915.1
3184
AAAACAUUGGUUUGGCACUAA
1073-1093
3480
UUAGUGCCAAACCAAUGUUUUUA
1071-1093

AD-239941.1
3185
GUCAAACGAACAAGAUUAAUU
1099-1119
3481
AAUUAAUCUUGUUCGUUUGACUG
1097-1119

AD-239944.1
3186
AAACGAACAAGAUUAAUUACA
1102-1122
3482
UGUAAUUAAUCUUGUUCGUUUGA
1100-1122

AD-239945.1
3187
AACGAACAAGAUUAAUUACCU
1103-1123
3483
AGGUAAUUAAUCUUGUUCGUUUG
1101-1123

AD-239946.1
3188
ACGAACAAGAUUAAUUACCUA
1104-1124
3484
UAGGUAAUUAAUCUUGUUCGUUU
1102-1124

AD-239947.1
3189
CGAACAAGAUUAAUUACCUGU
1105-1125
3485
ACAGGUAAUUAAUCUUGUUCGUU
1103-1125

AD-239977.1
3190
UCUCAAGAAUAUUUACGUAGU
1135-1155
3486
ACUACGUAAAUAUUCUUGAGAAA
1133-1155

AD-239979.1
3191
UCAAGAAUAUUUACGUAGUUU
1137-1157
3487
AAACUACGUAAAUAUUCUUGAGA
1135-1157

AD-239980.1
3192
CAAGAAUAUUUACGUAGUUUU
1138-1158
3488
AAAACUACGUAAAUAUUCUUGAG
1136-1158

AD-239998.1
3193
UUUCAUAGGUCUGUUUUUCCU
1157-1177
3489
AGGAAAAACAGACCUAUGAAAAA
1155-1177

AD-240019.1
3194
UUCAUGCCUCUUAAAAACUUA
1178-1198
3490
UAAGUUUUUAAGAGGCAUGAAAG
1176-1198

AD-240031.1
3195
AAAAACUUCUGUGCUUACAUA
1190-1210
3491
UAUGUAAGCACAGAAGUUUUUAA
1188-1210

AD-240032.1
3196
AAAACUUCUGUGCUUACAUAA
1191-1211
3492
UUAUGUAAGCACAGAAGUUUUUA
1189-1211

AD-240033.1
3197
AAACUUCUGUGCUUACAUAAA
1192-1212
3493
UUUAUGUAAGCACAGAAGUUUUU
1190-1212

AD-240064.1
3198
UUUUUCCAUUUAAAGGUGGAA
1245-1265
3494
UUCCACCUUUAAAUGGAAAAAUA
1243-1265

AD-240090.1
3199
CUACCUCCCUAAAAGUAAAUA
1271-1291
3495
UAUUUACUUUUAGGGAGGUAGCU
1269-1291

AD-240091.1
3200
UACCUCCCUAAAAGUAAAUAA
1272-1292
3496
UUAUUUACUUUUAGGGAGGUAGC
1270-1292

AD-240108.1
3201
UACAAAGAGAACUUAUUUACA
1290-1310
3497
UGUAAAUAAGUUCUCUUUGUAUU
1288-1310

AD-240117.1
3202
AACUUAUUUACACAGGGAAGA
1299-1319
3498
UCUUCCCUGUGUAAAUAAGUUCU
1297-1319

AD-240124.1
3203
UUACACAGGGAAGGUUUAAGA
1306-1326
3499
UCUUAAACCUUCCCUGUGUAAAU
1304-1326

AD-240125.1
3204
UACACAGGGAAGGUUUAAGAA
1307-1327
3500
UUCUUAAACCUUCCCUGUGUAAA
1305-1327

AD-240126.1
3205
ACACAGGGAAGGUUUAAGACU
1308-1328
3501
AGUCUUAAACCUUCCCUGUGUAA
1306-1328

AD-240135.1
3206
AGGUUUAAGACUGUUCAAGUA
1317-1337
3502
UACUUGAACAGUCUUAAACCUUC
1315-1337

AD-240138.1
3207
UUUAAGACUGUUCAAGUAGCA
1320-1340
3503
UGCUACUUGAACAGUCUUAAACC
1318-1340

AD-240140.1
3208
UAAGACUGUUCAAGUAGCAUU
1322-1342
3504
AAUGCUACUUGAACAGUCUUAAA
1320-1342

AD-240142.1
3209
AGACUGUUCAAGUAGCAUUCA
1324-1344
3505
UGAAUGCUACUUGAACAGUCUUA
1322-1344

AD-240145.1
3210
CUGUUCAAGUAGCAUUCCAAU
1327-1347
3506
AUUGGAAUGCUACUUGAACAGUC
1325-1347

AD-240147.1
3211
GUUCAAGUAGCAUUCCAAUCU
1329-1349
3507
AGAUUGGAAUGCUACUUGAACAG
1327-1349

AD-240148.1
3212
UUCAAGUAGCAUUCCAAUCUA
1330-1350
3508
UAGAUUGGAAUGCUACUUGAACA
1328-1350

AD-240154.1
3213
UAGCAUUCCAAUCUGUAGCCA
1336-1356
3509
UGGCUACAGAUUGGAAUGCUACU
1334-1356

AD-240155.1
3214
AGCAUUCCAAUCUGUAGCCAU
1337-1357
3510
AUGGCUACAGAUUGGAAUGCUAC
1335-1357

AD-240156.1
3215
GCAUUCCAAUCUGUAGCCAUA
1338-1358
3511
UAUGGCUACAGAUUGGAAUGCUA
1336-1358

AD-240171.1
3216
GCCAUGCCACAGAAUAUCAAA
1353-1373
3512
UUUGAUAUUCUGUGGCAUGGCUA
1351-1373

AD-240172.1
3217
CCAUGCCACAGAAUAUCAACA
1354-1374
3513
UGUUGAUAUUCUGUGGCAUGGCU
1352-1374

AD-240192.1
3218
AAGAACACAGAAUGAGUGCAA
1374-1394
3514
UUGCACUCAUUCUGUGUUCUUGU
1372-1394

AD-240227.1
3219
GUUUCAGCAGGCAGCUUUAUA
1409-1429
3515
UAUAAAGCUGCCUGCUGAAACUU
1407-1429

AD-240228.1
3220
UUUCAGCAGGCAGCUUUAUCU
1410-1430
3516
AGAUAAAGCUGCCUGCUGAAACU
1408-1430

AD-240237.1
3221
GCAGCUUUAUCUCAACCUGGA
1419-1439
3517
UCCAGGUUGAGAUAAAGCUGCCU
1417-1439

AD-240245.1
3222
AUCUCAACCUGGACAUAUUUU
1427-1447
3518
AAAAUAUGUCCAGGUUGAGAUAA
1425-1447

AD-240276.1
3223
AUUUGAAAGAUUUCCCUAGCA
1458-1478
3519
UGCUAGGGAAAUCUUUCAAAUGC
1456-1478

AD-240278.1
3224
UUGAAAGAUUUCCCUAGCCUA
1460-1480
3520
UAGGCUAGGGAAAUCUUUCAAAU
1458-1480

AD-240279.1
3225
UGAAAGAUUUCCCUAGCCUCU
1461-1481
3521
AGAGGCUAGGGAAAUCUUUCAAA
1459-1481

AD-240280.1
3226
GAAAGAUUUCCCUAGCCUCUU
1462-1482
3522
AAGAGGCUAGGGAAAUCUUUCAA
1460-1482

AD-240308.1
3227
CAUUAGCCCAAAACGGUGCAA
1490-1510
3523
UUGCACCGUUUUGGGCUAAUGAA
1488-1510

AD-240309.1
3228
AUUAGCCCAAAACGGUGCAAA
1491-1511
3524
UUUGCACCGUUUUGGGCUAAUGA
1489-1511

AD-240310.1
3229
UUAGCCCAAAACGGUGCAACU
1492-1512
3525
AGUUGCACCGUUUUGGGCUAAUG
1490-1512

AD-240311.1
3230
UAGCCCAAAACGGUGCAACUA
1493-1513
3526
UAGUUGCACCGUUUUGGGCUAAU
1491-1513

AD-240312.1
3231
AGCCCAAAACGGUGCAACUCU
1494-1514
3527
AGAGUUGCACCGUUUUGGGCUAA
1492-1514

AD-240313.1
3232
GCCCAAAACGGUGCAACUCUA
1495-1515
3528
UAGAGUUGCACCGUUUUGGGCUA
1493-1515

AD-240315.1
3233
CCAAAACGGUGCAACUCUAUU
1497-1517
3529
AAUAGAGUUGCACCGUUUUGGGC
1495-1517

AD-240317.1
3234
AAAACGGUGCAACUCUAUUCU
1499-1519
3530
AGAAUAGAGUUGCACCGUUUUGG
1497-1519

AD-240318.1
3235
AAACGGUGCAACUCUAUUCUA
1500-1520
3531
UAGAAUAGAGUUGCACCGUUUUG
1498-1520

AD-240319.1
3236
AACGGUGCAACUCUAUUCUGA
1501-1521
3532
UCAGAAUAGAGUUGCACCGUUUU
1499-1521

AD-240322.1
3237
GGUGCAACUCUAUUCUGGACU
1504-1524
3533
AGUCCAGAAUAGAGUUGCACCGU
1502-1524

AD-240324.1
3238
UGCAACUCUAUUCUGGACUUU
1506-1526
3534
AAAGUCCAGAAUAGAGUUGCACC
1504-1526

AD-240326.1
3239
CAACUCUAUUCUGGACUUUAU
1508-1528
3535
AUAAAGUCCAGAAUAGAGUUGCA
1506-1528

AD-240327.1
3240
AACUCUAUUCUGGACUUUAUU
1509-1529
3536
AAUAAAGUCCAGAAUAGAGUUGC
1507-1529

AD-240333.1
3241
AUUCUGGACUUUAUUACUUGA
1515-1535
3537
UCAAGUAAUAAAGUCCAGAAUAG
1513-1535

AD-240364.1
3242
UGUAUAACUCUGAAGUCCACA
1546-1566
3538
UGUGGACUUCAGAGUUAUACAGA
1544-1566

AD-240365.1
3243
GUAUAACUCUGAAGUCCACCA
1547-1567
3539
UGGUGGACUUCAGAGUUAUACAG
1545-1567

AD-240375.1
3244
GAAGUCCACCAAAAGUGGACA
1557-1577
3540
UGUCCACUUUUGGUGGACUUCAG
1555-1577

AD-240378.1
3245
GUCCACCAAAAGUGGACCCUA
1560-1580
3541
UAGGGUCCACUUUUGGUGGACUU
1558-1580

AD-240381.1
3246
CACCAAAAGUGGACCCUCUAU
1563-1583
3542
AUAGAGGGUCCACUUUUGGUGGA
1561-1583

AD-240382.1
3247
ACCAAAAGUGGACCCUCUAUA
1564-1584
3543
UAUAGAGGGUCCACUUUUGGUGG
1562-1584

AD-240383.1
3248
CCAAAAGUGGACCCUCUAUAU
1565-1585
3544
AUAUAGAGGGUCCACUUUUGGUG
1563-1585

AD-240385.1
3249
AAAAGUGGACCCUCUAUAUUU
1567-1587
3545
AAAUAUAGAGGGUCCACUUUUGG
1565-1587

AD-240386.1
3250
AAAGUGGACCCUCUAUAUUUA
1568-1588
3546
UAAAUAUAGAGGGUCCACUUUUG
1566-1588

AD-240387.1
3251
AAGUGGACCCUCUAUAUUUCA
1569-1589
3547
UGAAAUAUAGAGGGUCCACUUUU
1567-1589

AD-240419.1
3252
AUACAUUAUGAAAGGUGACCA
1612-1632
3548
UGGUCACCUUUCAUAAUGUAUCU
1610-1632

AD-240420.1
3253
UACAUUAUGAAAGGUGACCGA
1613-1633
3549
UCGGUCACCUUUCAUAAUGUAUC
1611-1633

AD-240421.1
3254
ACAUUAUGAAAGGUGACCGAA
1614-1634
3550
UUCGGUCACCUUUCAUAAUGUAU
1612-1634

AD-240422.1
3255
CAUUAUGAAAGGUGACCGACU
1615-1635
3551
AGUCGGUCACCUUUCAUAAUGUA
1613-1635

AD-240423.1
3256
AUUAUGAAAGGUGACCGACUA
1616-1636
3552
UAGUCGGUCACCUUUCAUAAUGU
1614-1636

AD-240431.1
3257
AGGUGACCGACUCUAUUUUAA
1624-1644
3553
UUAAAAUAGAGUCGGUCACCUUU
1622-1644

AD-240446.1
3258
AUCUCAGAAUUUUAAGUUCUA
1645-1665
3554
UAGAACUUAAAAUUCUGAGAUUU
1643-1665

AD-240474.1
3259
CUUUCAUAUAUCCUUGGUCCA
1699-1719
3555
UGGACCAAGGAUAUAUGAAAGCA
1697-1719

AD-240475.1
3260
UUUCAUAUAUCCUUGGUCCCA
1700-1720
3556
UGGGACCAAGGAUAUAUGAAAGC
1698-1720

AD-240476.1
3261
UUCAUAUAUCCUUGGUCCCAA
1701-1721
3557
UUGGGACCAAGGAUAUAUGAAAG
1699-1721

AD-240490.1
3262
GUCCCAGAGAUGUUUAGACAA
1715-1735
3558
UUGUCUAAACAUCUCUGGGACCA
1713-1735

AD-240494.1
3263
CAGAGAUGUUUAGACAAUUUU
1719-1739
3559
AAAAUUGUCUAAACAUCUCUGGG
1717-1739

AD-240495.1
3264
AGAGAUGUUUAGACAAUUUUA
1720-1740
3560
UAAAAUUGUCUAAACAUCUCUGG
1718-1740

AD-240501.1
3265
GUUUAGACAAUUUUAGGCUCA
1726-1746
3561
UGAGCCUAAAAUUGUCUAAACAU
1724-1746

AD-240502.1
3266
UUUAGACAAUUUUAGGCUCAA
1727-1747
3562
UUGAGCCUAAAAUUGUCUAAACA
1725-1747

AD-240503.1
3267
UUAGACAAUUUUAGGCUCAAA
1728-1748
3563
UUUGAGCCUAAAAUUGUCUAAAC
1726-1748

AD-240504.1
3268
UAGACAAUUUUAGGCUCAAAA
1729-1749
3564
UUUUGAGCCUAAAAUUGUCUAAA
1727-1749

AD-240505.1
3269
AGACAAUUUUAGGCUCAAAAA
1730-1750
3565
UUUUUGAGCCUAAAAUUGUCUAA
1728-1750

AD-240507.1
3270
ACAAUUUUAGGCUCAAAAAUU
1732-1752
3566
AAUUUUUGAGCCUAAAAUUGUCU
1730-1752

AD-240512.1
3271
UUUAGGCUCAAAAAUUAAAGA
1737-1757
3567
UCUUUAAUUUUUGAGCCUAAAAU
1735-1757

AD-240513.1
3272
UUAGGCUCAAAAAUUAAAGCU
1738-1758
3568
AGCUUUAAUUUUUGAGCCUAAAA
1736-1758

AD-240514.1
3273
UAGGCUCAAAAAUUAAAGCUA
1739-1759
3569
UAGCUUUAAUUUUUGAGCCUAAA
1737-1759

AD-240538.1
3274
CAGGAAAAGGAACUGUACUGA
1763-1783
3570
UCAGUACAGUUCCUUUUCCUGUG
1761-1783

AD-240540.1
3275
GGAAAAGGAACUGUACUGGCU
1765-1785
3571
AGCCAGUACAGUUCCUUUUCCUG
1763-1785

AD-240542.1
3276
AAAAGGAACUGUACUGGCUAU
1767-1787
3572
AUAGCCAGUACAGUUCCUUUUCC
1765-1787

AD-240545.1
3277
AGGAACUGUACUGGCUAUUAA
1770-1790
3573
UUAAUAGCCAGUACAGUUCCUUU
1768-1790

AD-240546.1
3278
GGAACUGUACUGGCUAUUACA
1771-1791
3574
UGUAAUAGCCAGUACAGUUCCUU
1769-1791

AD-240779.1
3279
UAAUCCGACUCCCACUACAUA
2181-2201
3575
UAUGUAGUGGGAGUCGGAUUAUU
2179-2201

AD-240782.1
3280
UCCGACUCCCACUACAUCAAA
2184-2204
3576
UUUGAUGUAGUGGGAGUCGGAUU
2182-2204

AD-240787.1
3281
CUCCCACUACAUCAAGACUAA
2189-2209
3577
UUAGUCUUGAUGUAGUGGGAGUC
2187-2209

AD-240788.1
3282
UCCCACUACAUCAAGACUAAU
2190-2210
3578
AUUAGUCUUGAUGUAGUGGGAGU
2188-2210

AD-240789.1
3283
CCCACUACAUCAAGACUAAUA
2191-2211
3579
UAUUAGUCUUGAUGUAGUGGGAG
2189-2211

AD-240790.1
3284
CCACUACAUCAAGACUAAUCU
2192-2212
3580
AGAUUAGUCUUGAUGUAGUGGGA
2190-2212

AD-240792.1
3285
ACUACAUCAAGACUAAUCUUA
2194-2214
3581
UAAGAUUAGUCUUGAUGUAGUGG
2192-2214

AD-240793.1
3286
CUACAUCAAGACUAAUCUUGU
2195-2215
3582
ACAAGAUUAGUCUUGAUGUAGUG
2193-2215

AD-240794.1
3287
UACAUCAAGACUAAUCUUGUU
2196-2216
3583
AACAAGAUUAGUCUUGAUGUAGU
2194-2216

AD-240795.1
3288
ACAUCAAGACUAAUCUUGUUU
2197-2217
3584
AAACAAGAUUAGUCUUGAUGUAG
2195-2217

AD-240826.1
3289
GUAUUAUAGAAUGCUUUUGCA
2234-2254
3585
UGCAAAAGCAUUCUAUAAUACAU
2232-2254

AD-240830.1
3290
UAUAGAAUGCUUUUGCAUGGA
2238-2258
3586
UCCAUGCAAAAGCAUUCUAUAAU
2236-2258

AD-240833.1
3291
AGAAUGCUUUUGCAUGGACUA
2241-2261
3587
UAGUCCAUGCAAAAGCAUUCUAU
2239-2261

AD-240834.1
3292
GAAUGCUUUUGCAUGGACUAU
2242-2262
3588
AUAGUCCAUGCAAAAGCAUUCUA
2240-2262

AD-240837.1
3293
UGCUUUUGCAUGGACUAUCCU
2245-2265
3589
AGGAUAGUCCAUGCAAAAGCAUU
2243-2265

AD-240839.1
3294
CUUUUGCAUGGACUAUCCUCU
2247-2267
3590
AGAGGAUAGUCCAUGCAAAAGCA
2245-2267

AD-240843.1
3295
UGCAUGGACUAUCCUCUUGUU
2251-2271
3591
AACAAGAGGAUAGUCCAUGCAAA
2249-2271

AD-240844.1
3296
GCAUGGACUAUCCUCUUGUUU
2252-2272
3592
AAACAAGAGGAUAGUCCAUGCAA
2250-2272

AD-240871.1
3297
AAAUAACCUCUUGUAGUUAUA
2349-2369
3593
UAUAACUACAAGAGGUUAUUUUU
2347-2369

AD-240875.1
3298
AACCUCUUGUAGUUAUAAAAU
2353-2373
3594
AUUUUAUAACUACAAGAGGUUAU
2351-2373

TABLE 3

HSD17B13 Modified Sequences

SEQ

SEQ

SEQ

ID
Sense Strand Sequence
ID

ID

Duplex Name
NO:
5′ to 3′
NO:
Strand Sequence 5′ to 3′
NO:
mRNA Target Sequence

AD-238911.1
3595
csasguacCfuCfCfUfcccu
3891
asGfsuccUfaGfGfgaggAfgGf
4187
GACAGUACCUCCUCCCUAGGACU

aggacuL96

uacugsusc

AD-238912.1
3596
asgsuaccUfcCfUfCfccua
3892
usAfsgucCfuAfGfggagGfaGf
4188
ACAGUACCUCCUCCCUAGGACUA

ggacuaL96

guacusgsu

AD-238913.1
3597
gsusaccuCfcUfCfCfcuag
3893
usUfsaguCfcUfAfgggaGfgAf
4189
CAGUACCUCCUCCCUAGGACUAC

gacuaaL96

gguacsusg

AD-238929.1
3598
ascsuacaCfaAfGfGfacug
3894
usUfsgguUfcAfGfuccuUfgUf
4190
GGACUACACAAGGACUGAACCAG

aaccaaL96

guaguscsc

AD-238930.1
3599
csusacacAfaGfGfAfcuga
3895
usCfsuggUfuCfAfguccUfuGf
4191
GACUACACAAGGACUGAACCAGA

accagaL96

uguagsusc

AD-238932.1
3600
ascsacaaGfgAfCfUfgaac
3896
usUfsucuGfgUfUfcaguCfcUf
4192
CUACACAAGGACUGAACCAGAAG

cagaaaL96

ugugusasg

AD-238960.1
3601
csasgagcAfaAfGfCfcaug
3897
usAfsuguUfcAfUfggcuUfuGf
4193
GACAGAGCAAAGCCAUGAACAUC

aacauaL96

cucugsusc

AD-238979.1
3602
uscsauccUfaGfAfAfaucc
3898
usCfsagaAfgGfAfuuucUfaGf
4194
CAUCAUCCUAGAAAUCCUUCUGC

uucugaL96

gaugasusg

AD-238991.1
3603
uscscuucUfgCfUfUfcuga
3899
usGfsgugAfuCfAfgaagCfaGf
4195
AAUCCUUCUGCUUCUGAUCACCA

ucaccaL96

aaggasusu

AD-239009.1
3604
cscsaucaUfcUfAfCfuccu
3900
usCfsaagUfaGfGfaguaGfaUf
4196
CACCAUCAUCUACUCCUACUUGG

acuugaL96

gauggsusg

AD-239015.1
3605
uscsuacuCfcUfAfCfuugg
3901
asCfsgacUfcCfAfaguaGfgAf
4197
CAUCUACUCCUACUUGGAGUCGU

agucguL96

guagasusg

AD-239016.1
3606
csusacucCfuAfCfUfugga
3902
asAfscgaCfuCfCfaaguAfgGf
4198
AUCUACUCCUACUUGGAGUCGUU

gucguuL96

aguagsasu

AD-239017.1
3607
usascuccUfaCfUfUfggag
3903
usAfsacgAfcUfCfcaagUfaGf
4199
UCUACUCCUACUUGGAGUCGUUG

ucguuaL96

gaguasgsa

AD-239018.1
3608
ascsuccuAfcUfUfGfgagu
3904
usCfsaacGfaCfUfccaaGfuAf
4200
CUACUCCUACUUGGAGUCGUUGG

cguugaL96

ggagusasg

AD-239019.1
3609
csusccuaCfuUfGfGfaguc
3905
asCfscaaCfgAfCfuccaAfgUf
4201
UACUCCUACUUGGAGUCGUUGGU

guugguL96

aggagsusa

AD-239020.1
3610
uscscuacUfuGfGfAfgucg
3906
usAfsccaAfcGfAfcuccAfaGf
4202
ACUCCUACUUGGAGUCGUUGGUG

uugguaL96

uaggasgsu

AD-239031.1
3611
gsuscguuGfgUfGfAfaguu
3907
asUfsgaaAfaAfCfuucaCfcAf
4203
GAGUCGUUGGUGAAGUUUUUCAU

uuucauL96

acgacsusc

AD-239062.1
3612
gsasgauuGfuUfCfUfcauu
3908
usCfscagUfaAfUfgagaAfcAf
4204
GGGAGAUUGUUCUCAUUACUGGA

acuggaL96

aucucscsc

AD-239066.1
3613
ususguucUfcAfUfUfacug
3909
usAfsgcuCfcAfGfuaauGfaGf
4205
GAUUGUUCUCAUUACUGGAGCUG

gagcuaL96

aacaasusc

AD-239103.1
3614
gscsagacUfaCfUfUfauga
3910
usCfsaaaUfuCfAfuaagUfaGf
4206
AGGCAGACUACUUAUGAAUUUGC

auuugaL96

ucugcscsu

AD-239110.1
3615
ascsuuauGfaAfUfUfugca
3911
usCfsguuUfuGfCfaaauUfcAf
4207
CUACUUAUGAAUUUGCAAAACGA

aaacgaL96

uaagusasg

AD-239118.1
3616
asusuugcAfaAfAfCfgaca
3912
asUfsgcuCfuGfUfcguuUfuGf
4208
GAAUUUGCAAAACGACAGAGCAU

gagcauL96

caaaususc

AD-239119.1
3617
ususugcaAfaAfCfGfacag
3913
usAfsugcUfcUfGfucguUfuUf
4209
AAUUUGCAAAACGACAGAGCAUA

agcauaL96

gcaaasusu

AD-239131.1
3618
csasgagcAfuAfUfUfgguu
3914
usCfsacaGfaAfCfcaauAfuGf
4210
GACAGAGCAUAUUGGUUCUGUGG

cugugaL96

cucugsusc

AD-239132.1
3619
asgsagcaUfaUfUfGfguuc
3915
usCfscacAfgAfAfccaaUfaUf
4211
ACAGAGCAUAUUGGUUCUGUGGG

uguggaL96

gcucusgsu

AD-239133.1
3620
gsasgcauAfuUfGfGfuucu
3916
usCfsccaCfaGfAfaccaAfuAf
4212
CAGAGCAUAUUGGUUCUGUGGGA

gugggaL96

ugcucsusg

AD-239146.1
3621
csusguggGfaUfAfUfuaau
3917
usCfsgcuUfaUfUfaauaUfcCf
4213
UUCUGUGGGAUAUUAAUAAGCGC

aagcgaL96

cacagsasa

AD-239155.1
3622
asusuaauAfaGfCfGfcggu
3918
usUfsccaCfaCfCfgcgcUfuAf
4214
AUAUUAAUAAGCGCGGUGUGGAG

guggaaL96

uuaausasu

AD-239156.1
3623
ususaauaAfgCfGfCfggug
3919
usCfsuccAfcAfCfcgcgCfuUf
4215
UAUUAAUAAGCGCGGUGUGGAGG

uggagaL96

auuaasusa

AD-239177.1
3624
asasacugCfaGfCfUfgagu
3920
usUfscggCfaCfUfcagcUfgCf
4216
GGAAACUGCAGCUGAGUGCCGAA

gccgaaL96

aguuuscsc

AD-239178.1
3625
asascugcAfgCfUfGfagug
3921
usUfsucgGfcAfCfucagCfuGf
4217
GAAACUGCAGCUGAGUGCCGAAA

ccgaaaL96

caguususc

AD-239179.1
3626
ascsugcaGfcUfGfAfgugc
3922
usUfsuucGfgCfAfcucaGfcUf
4218
AAACUGCAGCUGAGUGCCGAAAA

cgaaaaL96

gcagususu

AD-239180.1
3627
csusgcagCfuGfAfGfugcc
3923
gsUfsuuuCfgGfCfacucAfgCf
4219
AACUGCAGCUGAGUGCCGAAAAC

gaaaacL96

ugcagsusu

AD-239181.1
3628
usgscagcUfgAfGfUfgccg
3924
asGfsuuuUfcGfGfcacuCfaGf
4220
ACUGCAGCUGAGUGCCGAAAACU

aaaacuL96

cugcasgsu

AD-239182.1
3629
gscsagcuGfaGfUfGfccga
3925
usAfsguuUfuCfGfgcacUfcAf
4221
CUGCAGCUGAGUGCCGAAAACUA

aaacuaL96

gcugcsasg

AD-239183.1
3630
csasgcugAfgUfGfCfcgaa
3926
usUfsaguUfuUfCfggcaCfuCf
4222
UGCAGCUGAGUGCCGAAAACUAG

aacuaaL96

agcugscsa

AD-239185.1
3631
gscsugagUfgCfCfGfaaaa
3927
usCfscuaGfuUfUfucggCfaCf
4223
CAGCUGAGUGCCGAAAACUAGGC

cuaggaL96

ucagcsusg

AD-239186.1
3632
csusgaguGfcCfGfAfaaac
3928
usGfsccuAfgUfUfuucgGfcAf
4224
AGCUGAGUGCCGAAAACUAGGCG

uaggcaL96

cucagscsu

AD-239187.1
3633
usgsagugCfcGfAfAfaacu
3929
asCfsgccUfaGfUfuuucGfgCf
4225
GCUGAGUGCCGAAAACUAGGCGU

aggcguL96

acucasgsc

AD-239188.1
3634
gsasgugcCfgAfAfAfacua
3930
usAfscgcCfuAfGfuuuuCfgGf
4226
CUGAGUGCCGAAAACUAGGCGUC

ggcguaL96

cacucsasg

AD-239189.1
3635
asgsugccGfaAfAfAfcuag
3931
usGfsacgCfcUfAfguuuUfcGf
4227
UGAGUGCCGAAAACUAGGCGUCA

gcgucaL96

gcacuscsa

AD-239190.1
3636
gsusgccgAfaAfAfCfuagg
3932
usUfsgacGfcCfUfaguuUfuCf
4228
GAGUGCCGAAAACUAGGCGUCAC

cgucaaL96

ggcacsusc

AD-239191.1
3637
usgsccgaAfaAfCfUfaggc
3933
asGfsugaCfgCfCfuaguUfuUf
4229
AGUGCCGAAAACUAGGCGUCACU

gucacuL96

cggcascsu

AD-239192.1
3638
gscscgaaAfaCfUfAfggcg
3934
usAfsgugAfcGfCfcuagUfuUf
4230
GUGCCGAAAACUAGGCGUCACUG

ucacuaL96

ucggcsasc

AD-239193.1
3639
cscsgaaaAfcUfAfGfgcgu
3935
usCfsaguGfaCfGfccuaGfuUf
4231
UGCCGAAAACUAGGCGUCACUGC

cacugaL96

uucggscsa

AD-239194.1
3640
csgsaaaaCfuAfGfGfcguc
3936
usGfscagUfgAfCfgccuAfgUf
4232
GCCGAAAACUAGGCGUCACUGCG

acugcaL96

uuucgsgsc

AD-239196.1
3641
asasaacuAfgGfCfGfucac
3937
usGfscgcAfgUfGfacgcCfuAf
4233
CGAAAACUAGGCGUCACUGCGCA

ugcgcaL96

guuuuscsg

AD-239198.1
3642
asascuagGfcGfUfCfacug
3938
usAfsugcGfcAfGfugacGfcCf
4234
AAAACUAGGCGUCACUGCGCAUG

cgcauaL96

uaguususu

AD-239199.1
3643
ascsuaggCfgUfCfAfcugc
3939
usCfsaugCfgCfAfgugaCfgCf
4235
AAACUAGGCGUCACUGCGCAUGC

gcaugaL96

cuagususu

AD-239203.1
3644
gsgscgucAfcUfGfCfgcau
3940
asUfsacgCfaUfGfcgcaGfuGf
4236
UAGGCGUCACUGCGCAUGCGUAU

gcguauL96

acgccsusa

AD-239214.1
3645
gscsaugcGfuAfUfGfuggu
3941
usAfsgucUfaCfCfacauAfcGf
4237
GCGCAUGCGUAUGUGGUAGACUG

agacuaL96

caugcsgsc

AD-239215.1
3646
csasugcgUfaUfGfUfggua
3942
usCfsaguCfuAfCfcacaUfaCf
4238
CGCAUGCGUAUGUGGUAGACUGC

gacugaL96

gcaugscsg

AD-239239.1
3647
asascagaGfaAfGfAfgauc
3943
usCfsgauAfgAfUfcucuUfcUf
4239
GCAACAGAGAAGAGAUCUAUCGC

uaucgaL96

cuguusgsc

AD-239240.1
3648
ascsagagAfaGfAfGfaucu
3944
asGfscgaUfaGfAfucucUfuCf
4240
CAACAGAGAAGAGAUCUAUCGCU

aucgcuL96

ucugususg

AD-239241.1
3649
csasgagaAfgAfGfAfucua
3945
usAfsgcgAfuAfGfaucuCfuUf
4241
AACAGAGAAGAGAUCUAUCGCUC

ucgcuaL96

cucugsusu

AD-239242.1
3650
asgsagaaGfaGfAfUfcuau
3946
asGfsagcGfaUfAfgaucUfalf
4242
ACAGAGAAGAGAUCUAUCGCUCU

cgcucuL96

ucucusgsu

AD-239243.1
3651
gsasgaagAfgAfUfCfuauc
3947
usAfsgagCfgAfUfagauCfuCf
4243
CAGAGAAGAGAUCUAUCGCUCUC

gcucuaL96

uucucsusg

AD-239245.1
3652
gsasagagAfuCfUfAfucgc
3948
usAfsgagAfgCfGfauagAfuCf
4244
GAGAAGAGAUCUAUCGCUCUCUA

ucucuaL96

ucuucsusc

AD-239246.1
3653
asasgagaUfcUfAfUfcgcu
3949
usUfsagaGfaGfCfgauaGfaUf
4245
AGAAGAGAUCUAUCGCUCUCUAA

cucuaaL96

cucuuscsu

AD-239247.1
3654
asgsagauCfuAfUfCfgcuc
3950
usUfsuagAfgAfGfcgauAfgAf
4246
GAAGAGAUCUAUCGCUCUCUAAA

ucuaaaL96

ucucususc

AD-239250.1
3655
gsasucuaUfcGfCfUfcucu
3951
usGfsauuUfaGfAfgagcGfaUf
4247
GAGAUCUAUCGCUCUCUAAAUCA

aaaucaL96

agaucsusc

AD-239251.1
3656
asuscuauCfgCfUfCfucua
3952
usUfsgauUfuAfGfagagCfgAf
4248
AGAUCUAUCGCUCUCUAAAUCAG

aaucaaL96

uagauscsu

AD-239253.1
3657
csusaucgCfuCfUfCfuaaa
3953
asCfscugAfuUfUfagagAfgCf
4249
AUCUAUCGCUCUCUAAAUCAGGU

ucagguL96

gauagsasu

AD-239254.1
3658
usasucgcUfcUfCfUfaaau
3954
usAfsccuGfaUfUfuagaGfaGf
4250
UCUAUCGCUCUCUAAAUCAGGUG

cagguaL96

cgauasgsa

AD-239255.1
3659
asuscgcuCfuCfUfAfaauc
3955
usCfsaccUfgAfUfuuagAfgAf
4251
CUAUCGCUCUCUAAAUCAGGUGA

aggugaL96

gcgausasg

AD-239290.1
3660
gsasuguaAfcAfAfUfcgug
3956
asUfsucaCfcAfCfgauuGfuUf
4252
GUGAUGUAACAAUCGUGGUGAAU

gugaauL96

acaucsasc

AD-239291.1
3661
asusguaaCfaAfUfCfgugg
3957
usAfsuucAfcCfAfcgauUfgUf
4253
UGAUGUAACAAUCGUGGUGAAUA

ugaauaL96

uacauscsa

AD-239292.1
3662
usgsuaacAfaUfCfGfuggu
3958
usUfsauuCfaCfCfacgaUfuGf
4254
GAUGUAACAAUCGUGGUGAAUAA

gaauaaL96

uuacasusc

AD-239293.1
3663
gsusaacaAfuCfGfUfggug
3959
asUfsuauUfcAfCfcacgAfuUf
4255
AUGUAACAAUCGUGGUGAAUAAU

aauaauL96

guuacsasu

AD-239304.1
3664
gsgsugaaUfaAfUfGfcugg
3960
asCfsuguCfcCfAfgcauUfaUf
4256
GUGGUGAAUAAUGCUGGGACAGU

gacaguL96

ucaccsasc

AD-239312.1
3665
asusgcugGfgAfCfAfguau
3961
usUfsggaUfaUfAfcuguCfcCf
4257
UAAUGCUGGGACAGUAUAUCCAG

auccaaL96

agcaususa

AD-239313.1
3666
usgscuggGfaCfAfGfuaua
3962
usCfsuggAfuAfUfacugUfcCf
4258
AAUGCUGGGACAGUAUAUCCAGC

uccagaL96

cagcasusu

AD-239314.1
3667
gscsugggAfcAfGfUfauau
3963
usGfscugGfaUfAfuacuGfuCf
4259
AUGCUGGGACAGUAUAUCCAGCC

ccagcaL96

ccagcsasu

AD-239316.1
3668
usgsggacAfgUfAfUfaucc
3964
usCfsggcUfgGfAfuauaCfuGf
4260
GCUGGGACAGUAUAUCCAGCCGA

agccgaL96

ucccasgsc

AD-239317.1
3669
gsgsgacaGfuAfUfAfucca
3965
asUfscggCfuGfGfauauAfcUf
4261
CUGGGACAGUAUAUCCAGCCGAU

gccgauL96

gucccsasg

AD-239318.1
3670
gsgsacagUfaUfAfUfccag
3966
usAfsucgGfcUfGfgauaUfaCf
4262
UGGGACAGUAUAUCCAGCCGAUC

ccgauaL96

uguccscsa

AD-239321.1
3671
csasguauAfuCfCfAfgccg
3967
usAfsagaUfcGfGfcuggAfuAf
4263
GACAGUAUAUCCAGCCGAUCUUC

aucuuaL96

uacugsusc

AD-239332.1
3672
gscscgauCfuUfCfUfcagc
3968
usUfsuggUfgCfUfgagaAfgAf
4264
CAGCCGAUCUUCUCAGCACCAAG

accaaaL96

ucggcsusg

AD-239337.1
3673
uscsuucuCfaGfCfAfccaa
3969
usCfsaucCfuUfGfgugcUfgAf
4265
GAUCUUCUCAGCACCAAGGAUGA

ggaugaL96

gaagasusc

AD-239348.1
3674
cscsaaggAfuGfAfAfgaga
3970
usGfsguaAfuCfUfcuucAfuCf
4266
CACCAAGGAUGAAGAGAUUACCA

uuaccaL96

cuuggsusg

AD-239362.1
3675
asusuaccAfaGfAfCfauuu
3971
usAfsccuCfaAfAfugucUfuGf
4267
AGAUUACCAAGACAUUUGAGGUC

gagguaL96

guaauscsu

AD-239366.1
3676
cscsaagaCfaUfUfUfgagg
3972
usGfsuugAfcCfUfcaaaUfgUf
4268
UACCAAGACAUUUGAGGUCAACA

ucaacaL96

cuuggsusa

AD-239368.1
3677
asasgacaUfuUfGfAfgguc
3973
usAfsuguUfgAfCfcucaAfaUf
4269
CCAAGACAUUUGAGGUCAACAUC

aacauaL96

gucuusgsg

AD-239372.1
3678
csasuuugAfgGfUfCfaaca
3974
usUfsaggAfuGfUfugacCfuCf
4270
GACAUUUGAGGUCAACAUCCUAG

uccuaaL96

aaaugsusc

AD-239373.1
3679
asusuugaGfgUfCfAfacau
3975
usCfsuagGfaUfGfuugaCfcUf
4271
ACAUUUGAGGUCAACAUCCUAGG

ccuagaL96

caaausgsu

AD-239379.1
3680
gsgsucaaCfaUfCfCfuagg
3976
asAfsaugUfcCfUfaggaUfgUf
4272
GAGGUCAACAUCCUAGGACAUUU

acauuuL96

ugaccsusc

AD-239380.1
3681
gsuscaacAfuCfCfUfagga
3977
asAfsaauGfuCfCfuaggAfuGf
4273
AGGUCAACAUCCUAGGACAUUUU

cauuuuL96

uugacscsu

AD-239387.1
3682
uscscuagGfaCfAfUfuuuu
3978
usGfsaucCfaAfAfaaugUfcCf
4274
CAUCCUAGGACAUUUUUGGAUCA

ggaucaL96

uaggasusg

AD-239397.1
3683
ususuuugGfaUfCfAfcaaa
3979
asGfsugcUfuUfUfgugaUfcCf
4275
CAUUUUUGGAUCACAAAAGCACU

agcacuL96

aaaaasusg

AD-239411.1
3684
asasgcacUfuCfUfUfccau
3980
usCfsaucGfaUfGfgaagAfaGf
4276
AAAAGCACUUCUUCCAUCGAUGA

cgaugaL96

ugcuususu

AD-239412.1
3685
asgscacuUfcUfUfCfcauc
3981
asUfscauCfgAfUfggaaGfaAf
4277
AAAGCACUUCUUCCAUCGAUGAU

gaugauL96

gugcususu

AD-239431.1
3686
asusggagAfgAfAfAfucau
3982
usUfsggcCfaUfGfauuuCfuCf
4278
UGAUGGAGAGAAAUCAUGGCCAC

ggccaaL96

uccauscsa

AD-239433.1
3687
gsgsagagAfaAfUfCfaugg
3983
asUfsgugGfcCfAfugauUfuCf
4279
AUGGAGAGAAAUCAUGGCCACAU

ccacauL96

ucuccsasu

AD-239436.1
3688
gsasgaaaUfcAfUfGfgcca
3984
asCfsgauGfuGfGfccauGfaUf
4280
GAGAGAAAUCAUGGCCACAUCGU

caucguL96

uucucsusc

AD-239437.1
3689
asgsaaauCfaUfGfGfccac
3985
usAfscgaUfgUfGfgccaUfgAf
4281
AGAGAAAUCAUGGCCACAUCGUC

aucguaL96

uuucuscsu

AD-239438.1
3690
gsasaaucAfuGfGfCfcaca
3986
usGfsacgAfuGfUfggccAfuGf
4282
GAGAAAUCAUGGCCACAUCGUCA

ucgucaL96

auuucsusc

AD-239439.1
3691
asasaucaUfgGfCfCfacau
3987
usUfsgacGfaUfGfuggcCfaUf
4283
AGAAAUCAUGGCCACAUCGUCAC

cgucaaL96

gauuuscsu

AD-239450.1
3692
ascsaucgUfcAfCfAfgugg
3988
usUfsgaaGfcCfAfcuguGfaCf
4284
CCACAUCGUCACAGUGGCUUCAG

cuucaaL96

gaugusgsg

AD-239451.1
3693
csasucguCfaCfAfGfuggc
3989
asCfsugaAfgCfCfacugUfgAf
4285
CACAUCGUCACAGUGGCUUCAGU

uucaguL96

cgaugsusg

AD-239452.1
3694
asuscgucAfcAfGfUfggcu
3990
usAfscugAfaGfCfcacuGfuGf
4286
ACAUCGUCACAGUGGCUUCAGUG

ucaguaL96

acgausgsu

AD-239453.1
3695
uscsgucaCfaGfUfGfgcuu
3991
asCfsacuGfaAfGfccacUfgUf
4287
CAUCGUCACAGUGGCUUCAGUGU

caguguL96

gacgasusg

AD-239475.1
3696
csgsgccaCfgAfAfGfggau
3992
usAfsaggAfaUfCfccuuCfgUf
4288
UGCGGCCACGAAGGGAUUCCUUA

uccuuaL96

ggccgscsa

AD-239476.1
3697
gsgsccacGfaAfGfGfgauu
3993
usUfsaagGfaAfUfcccuUfcGf
4289
GCGGCCACGAAGGGAUUCCUUAC

ccuuaaL96

uggccsgsc

AD-239478.1
3698
cscsacgaAfgGfGfAfuucc
3994
asGfsguaAfgGfAfauccCfuUf
4290
GGCCACGAAGGGAUUCCUUACCU

uuaccuL96

cguggscsc

AD-239479.1
3699
csascgaaGfgGfAfUfuccu
3995
usAfsgguAfaGfGfaaucCfcUf
4291
GCCACGAAGGGAUUCCUUACCUC

uaccuaL96

ucgugsgsc

AD-239480.1
3700
ascsgaagGfgAfUfUfccuu
3996
usGfsaggUfaAfGfgaauCfcCf
4292
CCACGAAGGGAUUCCUUACCUCA

accucaL96

uucgusgsg

AD-239482.1
3701
gsasagggAfuUfCfCfuuac
3997
usAfsugaGfgUfAfaggaAfuCf
4293
ACGAAGGGAUUCCUUACCUCAUC

cucauaL96

ccuucsgsu

AD-239483.1
3702
asasgggaUfuCfCfUfuacc
3998
usGfsaugAfgGfUfaaggAfaUf
4294
CGAAGGGAUUCCUUACCUCAUCC

ucaucaL96

cccuuscsg

AD-239499.1
3703
csasucccAfuAfUfUfguuc
3999
usUfsgcuGfgAfAfcaauAfuGf
4295
CUCAUCCCAUAUUGUUCCAGCAA

cagcaaL96

ggaugsasg

AD-239510.1
3704
gsusuccaGfcAfAfAfuuug
4000
usAfsgcgGfcAfAfauuuGfcUf
4296
UUGUUCCAGCAAAUUUGCCGCUG

ccgcuaL96

ggaacsasa

AD-239520.1
3705
asusuugcCfgCfUfGfuugg
4001
usGfsaaaGfcCfAfacagCfgGf
4297
AAAUUUGCCGCUGUUGGCUUUCA

cuuucaL96

caaaususu

AD-239521.1
3706
ususugccGfcUfGfUfuggc
4002
usUfsgaaAfgCfCfaacaGfcGf
4298
AAUUUGCCGCUGUUGGCUUUCAC

uuucaaL96

gcaaasusu

AD-239522.1
3707
ususgccgCfuGfUfUfggcu
4003
usGfsugaAfaGfCfcaacAfgCf
4299
AUUUGCCGCUGUUGGCUUUCACA

uucacaL96

ggcaasasu

AD-239534.1
3708
gscsuuucAfcAfGfAfgguc
4004
asUfsgucAfgAfCfcucuGfuGf
4300
UGGCUUUCACAGAGGUCUGACAU

ugacauL96

aaagcscsa

AD-239539.1
3709
csascagaGfgUfCfUfgaca
4005
usUfscugAfuGfUfcagaCfcUf
4301
UUCACAGAGGUCUGACAUCAGAA

ucagaaL96

cugugsasa

AD-239544.1
3710
asgsgucuGfaCfAfUfcaga
4006
usGfsaagUfuCfUfgaugUfcAf
4302
AGAGGUCUGACAUCAGAACUUCA

acuucaL96

gaccuscsu

AD-239566.1
3711
gscscuugGfgAfAfAfaacu
4007
usAfsuacCfaGfUfuuuuCfcCf
4303
AGGCCUUGGGAAAAACUGGUAUC

gguauaL96

aaggcscsu

AD-239578.1
3712
ascsugguAfuCfAfAfaacc
4008
asCfsaugAfgGfUfuuugAfuAf
4304
AAACUGGUAUCAAAACCUCAUGU

ucauguL96

ccagususu

AD-239579.1
3713
csusgguaUfcAfAfAfaccu
4009
usAfscauGfaGfGfuuuuGfaUf
4305
AACUGGUAUCAAAACCUCAUGUC

cauguaL96

accagsusu

AD-239611.1
3714
ususugugAfaUfAfCfuggg
4010
usGfsugaAfcCfCfaguaUfuCf
4306
UUUUUGUGAAUACUGGGUUCACC

uucacaL96

acaaasasa

AD-239612.1
3715
ususgugaAfuAfCfUfgggu
4011
usGfsgugAfaCfCfcaguAfuUf
4307
UUUUGUGAAUACUGGGUUCACCA

ucaccaL96

cacaasasa

AD-239613.1
3716
usgsugaaUfaCfUfGfgguu
4012
usUfsgguGfaAfCfccagUfaUf
4308
UUUGUGAAUACUGGGUUCACCAA

caccaaL96

ucacasasa

AD-239617.1
3717
asasuacuGfgGfUfUfcacc
4013
asUfsuuuUfgGfUfgaacCfcAf
4309
UGAAUACUGGGUUCACCAAAAAU

aaaaauL96

guauuscsa

AD-239618.1
3718
asusacugGfgUfUfCfacca
4014
usAfsuuuUfuGfGfugaaCfcCf
4310
GAAUACUGGGUUCACCAAAAAUC

aaaauaL96

aguaususc

AD-239637.1
3719
uscscaagCfaCfAfAfgauu
4015
usGfsccaUfaAfUfcuugUfgCf
4311
AAUCCAAGCACAAGAUUAUGGCC

auggcaL96

uuggasusu

AD-239641.1
3720
asgscacaAfgAfUfUfaugg
4016
usAfscagGfcCfAfuaauCfuUf
4312
CAAGCACAAGAUUAUGGCCUGUA

ccuguaL96

gugcususg

AD-239642.1
3721
gscsacaaGfaUfUfAfuggc
4017
asUfsacaGfgCfCfauaaUfcUf
4313
AAGCACAAGAUUAUGGCCUGUAU

cuguauL96

ugugcsusu

AD-239643.1
3722
csascaagAfuUfAfUfggcc
4018
asAfsuacAfgGfCfcauaAfuCf
4314
AGCACAAGAUUAUGGCCUGUAUU

uguauuL96

uugugscsu

AD-239645.1
3723
csasagauUfaUfGfGfccug
4019
usCfsaauAfcAfGfgccaUfaAf
4315
CACAAGAUUAUGGCCUGUAUUGG

uauugaL96

ucuugsusg

AD-239647.1
3724
asgsauuaUfgGfCfCfugua
4020
usUfsccaAfuAfCfaggcCfaUf
4316
CAAGAUUAUGGCCUGUAUUGGAG

uuggaaL96

aaucususg

AD-239649.1
3725
asusuaugGfcCfUfGfuauu
4021
usUfscucCfaAfUfacagGfcCf
4317
AGAUUAUGGCCUGUAUUGGAGAC

ggagaaL96

auaauscsu

AD-239650.1
3726
ususauggCfcUfGfUfauug
4022
usGfsucuCfcAfAfuacaGfgCf
4318
GAUUAUGGCCUGUAUUGGAGACA

gagacaL96

cauaasusc

AD-239653.1
3727
usgsgccuGfuAfUfUfggag
4023
asUfscugUfcUfCfcaauAfcAf
4319
UAUGGCCUGUAUUGGAGACAGAU

acagauL96

ggccasusa

AD-239654.1
3728
gsgsccugUfaUfUfGfgaga
4024
usAfsucuGfuCfUfccaaUfaCf
4320
AUGGCCUGUAUUGGAGACAGAUG

cagauaL96

aggccsasu

AD-239662.1
3729
ususggagAfcAfGfAfugaa
4025
usAfscgaCfuUfCfaucuGfuCf
4321
UAUUGGAGACAGAUGAAGUCGUA

gucguaL96

uccaasusa

AD-239668.1
3730
ascsagauGfaAfGfUfcgua
4026
asCfsuucUfuAfCfgacuUfcAf
4322
AGACAGAUGAAGUCGUAAGAAGU

agaaguL96

ucuguscsu

AD-239669.1
3731
csasgaugAfaGfUfCfguaa
4027
usAfscuuCfuUfAfcgacUfuCf
4323
GACAGAUGAAGUCGUAAGAAGUC

gaaguaL96

aucugsusc

AD-239670.1
3732
asgsaugaAfgUfCfGfuaag
4028
asGfsacuUfcUfUfacgaCfuUf
4324
ACAGAUGAAGUCGUAAGAAGUCU

aagucuL96

caucusgsu

AD-239671.1
3733
gsasugaaGfuCfGfUfaaga
4029
usAfsgacUfuCfUfuacgAfcUf
4325
CAGAUGAAGUCGUAAGAAGUCUG

agucuaL96

ucaucsusg

AD-239674.1
3734
gsasagucGfuAfAfGfaagu
4030
usAfsucaGfaCfUfucuuAfcGf
4326
AUGAAGUCGUAAGAAGUCUGAUA

cugauaL96

acuucsasu

AD-239676.1
3735
asgsucguAfaGfAfAfgucu
4031
usCfsuauCfaGfAfcuucUfuAf
4327
GAAGUCGUAAGAAGUCUGAUAGA

gauagaL96

cgacususc

AD-239681.1
3736
usasagaaGfuCfUfGfauag
4032
usUfsccaUfcUfAfucagAfcUf
4328
CGUAAGAAGUCUGAUAGAUGGAA

auggaaL96

ucuuascsg

AD-239682.1
3737
asasgaagUfcUfGfAfuaga
4033
asUfsuccAfuCfUfaucaGfaCf
4329
GUAAGAAGUCUGAUAGAUGGAAU

uggaauL96

uucuusasc

AD-239684.1
3738
gsasagucUfgAfUfAfgaug
4034
usUfsauuCfcAfUfcuauCfaGf
4330
AAGAAGUCUGAUAGAUGGAAUAC

gaauaaL96

acuucsusu

AD-239685.1
3739
asasgucuGfaUfAfGfaugg
4035
asGfsuauUfcCfAfucuaUfcAf
4331
AGAAGUCUGAUAGAUGGAAUACU

aauacuL96

gacuuscsu

AD-239697.1
3740
usgsgaauAfcUfUfAfccaa
4036
usUfscuuAfuUfGfguaaGfuAf
4332
GAUGGAAUACUUACCAAUAAGAA

uaagaaL96

uuccasusc

AD-239713.1
3741
asasugauUfuUfUfGfuucc
4037
usAfscgaUfgGfAfacaaAfaAf
4333
AAAAUGAUUUUUGUUCCAUCGUA

aucguaL96

ucauususu

AD-239714.1
3742
asusgauuUfuUfGfUfucca
4038
asUfsacgAfuGfGfaacaAfaAf
4334
AAAUGAUUUUUGUUCCAUCGUAU

ucguauL96

aucaususu

AD-239717.1
3743
asusuuuuGfuUfCfCfaucg
4039
usAfsuauAfcGfAfuggaAfcAf
4335
UGAUUUUUGUUCCAUCGUAUAUC

uauauaL96

aaaauscsa

AD-239721.1
3744
ususguucCfaUfCfGfuaua
4040
usAfsuugAfuAfUfacgaUfgGf
4336
UUUUGUUCCAUCGUAUAUCAAUA

ucaauaL96

aacaasasa

AD-239723.1
3745
gsusuccaUfcGfUfAfuauc
4041
usAfsuauUfgAfUfauacGfaUf
4337
UUGUUCCAUCGUAUAUCAAUAUC

aauauaL96

ggaacsasa

AD-239724.1
3746
ususccauCfgUfAfUfauca
4042
asGfsauaUfuGfAfuauaCfgAf
4338
UGUUCCAUCGUAUAUCAAUAUCU

auaucuL96

uggaascsa

AD-239725.1
3747
uscscaucGfuAfUfAfucaa
4043
asAfsgauAfuUfGfauauAfcGf
4339
GUUCCAUCGUAUAUCAAUAUCUU

uaucuuL96

auggasasc

AD-239726.1
3748
cscsaucgUfaUfAfUfcaau
4044
asAfsagaUfaUfUfgauaUfaCf
4340
UUCCAUCGUAUAUCAAUAUCUUU

aucuuuL96

gauggsasa

AD-239755.1
3749
ascsagaaGfuUfUfCfuucc
4045
usGfsuucAfgGfAfagaaAfcUf
4341
CUACAGAAGUUUCUUCCUGAACG

ugaacaL96

ucugusasg

AD-239757.1
3750
asgsaaguUfuCfUfUfccug
4046
usGfscguUfcAfGfgaagAfaAf
4342
ACAGAAGUUUCUUCCUGAACGCG

aacgcaL96

cuucusgsu

AD-239759.1
3751
asasguuuCfuUfCfCfugaa
4047
usGfscgcGfuUfCfaggaAfgAf
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AD-239905.1
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AD-239941.1
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AD-239998.1
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AD-240126.1
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AD-240135.1
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AD-240138.1
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AD-240140.1
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AD-240171.1
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AD-240172.1
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AD-240227.1
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AD-240279.1
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AD-240431.1
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AD-240474.1
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AD-240475.1
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ususucauAfuAfUfCfcuug
4148
usGfsggaCfcAfAfggauAfuAf
4444
GCUUUCAUAUAUCCUUGGUCCCA

gucccaL96

ugaaasgsc

AD-240476.1
3853
ususcauaUfaUfCfCfuugg
4149
usUfsgggAfcCfAfaggaUfaUf
4445
CUUUCAUAUAUCCUUGGUCCCAG

ucccaaL96

augaasasg

AD-240490.1
3854
gsuscccaGfaGfAfUfguuu
4150
usUfsgucUfaAfAfcaucUfcUf
4446
UGGUCCCAGAGAUGUUUAGACAA

agacaaL96

gggacscsa

AD-240494.1
3855
csasgagaUfgUfUfUfagac
4151
asAfsaauUfgUfCfuaaaCfaUf
4447
CCCAGAGAUGUUUAGACAAUUUU

aauuuuL96

cucugsgsg

AD-240495.1
3856
asgsagauGfuUfUfAfgaca
4152
usAfsaaaUfuGfUfcuaaAfcAf
4448
CCAGAGAUGUUUAGACAAUUUUA

auuuuaL96

ucucusgsg

AD-240501.1
3857
gsusuuagAfcAfAfUfuuua
4153
usGfsagcCfuAfAfaauuGfuCf
4449
AUGUUUAGACAAUUUUAGGCUCA

ggcucaL96

uaaacsasu

AD-240502.1
3858
ususuagaCfaAfUfUfuuag
4154
usUfsgagCfcUfAfaaauUfgUf
4450
UGUUUAGACAAUUUUAGGCUCAA

gcucaaL96

cuaaascsa

AD-240503.1
3859
ususagacAfaUfUfUfuagg
4155
usUfsugaGfcCfUfaaaaUfuGf
4451
GUUUAGACAAUUUUAGGCUCAAA

cucaaaL96

ucuaasasc

AD-240504.1
3860
usasgacaAfuUfUfUfaggc
4156
usUfsuugAfgCfCfuaaaAfuUf
4452
UUUAGACAAUUUUAGGCUCAAAA

ucaaaaL96

gucuasasa

AD-240505.1
3861
asgsacaaUfuUfUfAfggcu
4157
usUfsuuuGfaGfCfcuaaAfaUf
4453
UUAGACAAUUUUAGGCUCAAAAA

caaaaaL96

ugucusasa

AD-240507.1
3862
ascsaauuUfuAfGfGfcuca
4158
asAfsuuuUfuGfAfgccuAfaAf
4454
AGACAAUUUUAGGCUCAAAAAUU

aaaauuL96

auuguscsu

AD-240512.1
3863
ususuaggCfuCfAfAfaaau
4159
usCfsuuuAfaUfUfuuugAfgCf
4455
AUUUUAGGCUCAAAAAUUAAAGC

uaaagaL96

cuaaasasu

AD-240513.1
3864
ususaggcUfcAfAfAfaauu
4160
asGfscuuUfaAfUfuuuuGfaGf
4456
UUUUAGGCUCAAAAAUUAAAGCU

aaagcuL96

ccuaasasa

AD-240514.1
3865
usasggcuCfaAfAfAfauua
4161
usAfsgcuUfuAfAfuuuuUfgAf
4457
UUUAGGCUCAAAAAUUAAAGCUA

aagcuaL96

gccuasasa

AD-240538.1
3866
csasggaaAfaGfGfAfacug
4162
usCfsaguAfcAfGfuuccUfuUf
4458
CACAGGAAAAGGAACUGUACUGG

uacugaL96

uccugsusg

AD-240540.1
3867
gsgsaaaaGfgAfAfCfugua
4163
asGfsccaGfuAfCfaguuCfcUf
4459
CAGGAAAAGGAACUGUACUGGCU

cuggcuL96

uuuccsusg

AD-240542.1
3868
asasaaggAfaCfUfGfuacu
4164
asUfsagcCfaGfUfacagUfuCf
4460
GGAAAAGGAACUGUACUGGCUAU

ggcuauL96

cuuuuscsc

AD-240545.1
3869
asgsgaacUfgUfAfCfuggc
4165
usUfsaauAfgCfCfaguaCfaGf
4461
AAAGGAACUGUACUGGCUAUUAC

uauuaaL96

uuccususu

AD-240546.1
3870
gsgsaacuGfuAfCfUfggcu
4166
usGfsuaaUfaGfCfcaguAfcAf
4462
AAGGAACUGUACUGGCUAUUACA

auuacaL96

guuccsusu

AD-240779.1
3871
usasauccGfaCfUfCfccac
4167
usAfsuguAfgUfGfggagUfcGf
4463
AAUAAUCCGACUCCCACUACAUC

uacauaL96

gauuasusu

AD-240782.1
3872
uscscgacUfcCfCfAfcuac
4168
usUfsugaUfgUfAfguggGfaGf
4464
AAUCCGACUCCCACUACAUCAAG

aucaaaL96

ucggasusu

AD-240787.1
3873
csuscccaCfuAfCfAfucaa
4169
usUfsaguCfuUfGfauguAfgUf
4465
GACUCCCACUACAUCAAGACUAA

gacuaaL96

gggagsusc

AD-240788.1
3874
uscsccacUfaCfAfUfcaag
4170
asUfsuagUfcUfUfgaugUfaGf
4466
ACUCCCACUACAUCAAGACUAAU

acuaauL96

ugggasgsu

AD-240789.1
3875
cscscacuAfcAfUfCfaaga
4171
usAfsuuaGfuCfUfugauGfuAf
4467
CUCCCACUACAUCAAGACUAAUC

cuaauaL96

gugggsasg

AD-240790.1
3876
cscsacuaCfaUfCfAfagac
4172
asGfsauuAfgUfCfuugaUfgUf
4468
UCCCACUACAUCAAGACUAAUCU

uaaucuL96

aguggsgsa

AD-240792.1
3877
ascsuacaUfcAfAfGfacua
4173
usAfsagaUfuAfGfucuuGfaUf
4469
CCACUACAUCAAGACUAAUCUUG

aucuuaL96

guagusgsg

AD-240793.1
3878
csusacauCfaAfGfAfcuaa
4174
asCfsaagAfuUfAfgucuUfgAf
4470
CACUACAUCAAGACUAAUCUUGU

ucuuguL96

uguagsusg

AD-240794.1
3879
usascaucAfaGfAfCfuaau
4175
asAfscaaGfaUfUfagucUfuGf
4471
ACUACAUCAAGACUAAUCUUGUU

cuuguuL96

auguasgsu

AD-240795.1
3880
ascsaucaAfgAfCfUfaauc
4176
asAfsacaAfgAfUfuaguCfuUf
4472
CUACAUCAAGACUAAUCUUGUUU

uuguuuL96

gaugusasg

AD-240826.1
3881
gsusauuaUfaGfAfAfugcu
4177
usGfscaaAfaGfCfauucUfaUf
4473
AUGUAUUAUAGAAUGCUUUUGCA

uuugcaL96

aauacsasu

AD-240830.1
3882
usasuagaAfuGfCfUfuuug
4178
usCfscauGfcAfAfaagcAfuUf
4474
AUUAUAGAAUGCUUUUGCAUGGA

cauggaL96

cuauasasu

AD-240833.1
3883
asgsaaugCfuUfUfUfgcau
4179
usAfsgucCfaUfGfcaaaAfgCf
4475
AUAGAAUGCUUUUGCAUGGACUA

ggacuaL96

auucusasu

AD-240834.1
3884
gsasaugcUfuUfUfGfcaug
4180
asUfsaguCfcAfUfgcaaAfaGf
4476
UAGAAUGCUUUUGCAUGGACUAU

gacuauL96

cauucsusa

AD-240837.1
3885
usgscuuuUfgCfAfUfggac
4181
asGfsgauAfgUfCfcaugCfaAf
4477
AAUGCUUUUGCAUGGACUAUCCU

uauccuL96

aagcasusu

AD-240839.1
3886
csusuuugCfaUfGfGfacua
4182
asGfsaggAfuAfGfuccaUfgCf
4478
UGCUUUUGCAUGGACUAUCCUCU

uccucuL96

aaaagscsa

AD-240843.1
3887
usgscaugGfaCfUfAfuccu
4183
asAfscaaGfaGfGfauagUfcCf
4479
UUUGCAUGGACUAUCCUCUUGUU

cuuguuL96

augcasasa

AD-240844.1
3888
gscsauggAfcUfAfUfccuc
4184
asAfsacaAfgAfGfgauaGfuCf
4480
UUGCAUGGACUAUCCUCUUGUUU

uuguuuL96

caugcsasa

AD-240871.1
3889
asasauaaCfcUfCfUfugua
4185
usAfsuaaCfuAfCfaagaGfgUf
4481
AAAAAUAACCUCUUGUAGUUAUA

guuauaL96

uauuususu

AD-240875.1
3890
asasccucUfuGfUfAfguua
4186
asUfsuuuAfuAfAfcuacAfaGf
4482
AUAACCUCUUGUAGUUAUAAAAU

uaaaauL96

agguusasu

TABLE 4

HSD17B13 in vitro 50 nM screen in Cos-7

(Dual-Luciferase psiCHECK2 vector)

% of Human Message

Duplex Name
Remaining 50 nM
STDEV 50 nM

AD-238911.1
56.0
9.0

AD-238912.1
103.7
14.5

AD-238913.1
51.6
9.4

AD-238929.1
51.8
10.1

AD-238930.1
63.6
6.7

AD-238932.1
53.7
10.3

AD-238960.1
35.1
3.6

AD-238979.1
25.6
4.7

AD-238991.1
31.8
2.0

AD-239009.1
81.1
15.9

AD-239015.1
94.7
11.3

AD-239016.1
63.8
4.5

AD-239017.1
45.4
9.9

AD-239018.1
68.7
10.7

AD-239019.1
36.9
7.4

AD-239020.1
52.7
5.1

AD-239031.1
19.4
2.5

AD-239062.1
33.5
6.4

AD-239066.1
45.5
3.8

AD-239103.1
12.8
1.3

AD-239110.1
14.0
2.1

AD-239118.1
57.0
8.6

AD-239119.1
38.6
1.6

AD-239131.1
65.9
9.1

AD-239132.1
39.4
13.4

AD-239133.1
31.7
3.2

AD-239146.1
13.5
1.6

AD-239155.1
27.5
1.9

AD-239156.1
81.1
12.5

AD-239177.1
27.7
5.2

AD-239178.1
60.6
7.0

AD-239179.1
42.9
8.4

AD-239180.1
83.8
16.0

AD-239181.1
43.2
8.0

AD-239182.1
34.0
5.5

AD-239183.1
24.3
3.1

AD-239185.1
82.9
12.6

AD-239186.1
89.0
9.8

AD-239187.1
85.1
18.1

AD-239188.1
64.5
3.0

AD-239189.1
70.7
8.9

AD-239190.1
91.9
7.9

AD-239191.1
58.9
10.3

AD-239192.1
60.7
17.3

AD-239193.1
61.9
8.1

AD-239194.1
74.4
6.2

AD-239196.1
94.7
17.1

AD-239198.1
100.9
22.5

AD-239199.1
47.3
12.8

AD-239203.1
40.4
2.3

AD-239214.1
33.3
6.2

AD-239215.1
38.9
8.4

AD-239239.1
53.4
7.2

AD-239240.1
27.8
1.8

AD-239241.1
51.4
11.1

AD-239242.1
17.8
2.1

AD-239243.1
54.1
12.2

AD-239245.1
51.6
9.6

AD-239246.1
21.8
4.1

AD-239247.1
30.9
5.0

AD-239250.1
54.2
8.4

AD-239251.1
15.9
6.0

AD-239253.1
22.4
5.0

AD-239254.1
47.2
15.2

AD-239255.1
49.1
5.7

AD-239290.1
31.4
8.3

AD-239291.1
27.9
8.6

AD-239292.1
30.2
4.2

AD-239293.1
51.8
16.1

AD-239304.1
61.4
11.8

AD-239312.1
99.1
12.9

AD-239313.1
95.8
5.0

AD-239314.1
84.2
4.7

AD-239316.1
52.2
2.2

AD-239317.1
27.7
4.6

AD-239318.1
33.6
8.4

AD-239321.1
37.5
3.1

AD-239332.1
28.5
5.9

AD-239337.1
52.0
17.0

AD-239348.1
26.0
4.3

AD-239362.1
49.7
7.9

AD-239366.1
68.4
10.0

AD-239368.1
26.9
3.5

AD-239372.1
48.0
6.3

AD-239373.1
50.8
12.0

AD-239379.1
21.5
6.9

AD-239380.1
18.1
3.2

AD-239387.1
16.6
1.6

AD-239397.1
15.3
2.3

AD-239411.1
46.6
12.2

AD-239412.1
76.0
19.4

AD-239431.1
60.2
8.3

AD-239433.1
58.5
11.7

AD-239436.1
77.6
5.7

AD-239437.1
46.9
6.2

AD-239438.1
89.3
27.8

AD-239439.1
61.7
13.6

AD-239450.1
67.4
4.5

AD-239451.1
38.9
7.3

AD-239452.1
84.0
17.0

AD-239453.1
69.1
4.1

AD-239475.1
77.3
13.1

AD-239476.1
67.4
15.1

AD-239478.1
79.3
6.2

AD-239479.1
58.0
4.6

AD-239480.1
26.6
2.8

AD-239482.1
24.3
5.8

AD-239483.1
39.7
8.7

AD-239499.1
53.4
11.4

AD-239510.1
64.1
7.5

AD-239520.1
21.1
3.2

AD-239521.1
26.4
7.7

AD-239522.1
34.2
4.4

AD-239534.1
64.5
14.1

AD-239539.1
68.8
10.5

AD-239544.1
48.5
10.2

AD-239566.1
43.3
5.3

AD-239578.1
19.3
1.8

AD-239579.1
67.0
7.5

AD-239611.1
43.1
10.0

AD-239612.1
54.7
2.8

AD-239613.1
69.7
12.4

AD-239617.1
108.1
8.3

AD-239618.1
32.5
8.9

AD-239637.1
29.8
7.2

AD-239641.1
52.6
8.7

AD-239642.1
11.4
2.0

AD-239643.1
17.2
5.2

AD-239645.1
30.5
8.5

AD-239647.1
33.6
4.5

AD-239649.1
38.4
2.2

AD-239650.1
79.5
16.5

AD-239653.1
42.8
7.3

AD-239654.1
74.4
7.0

AD-239662.1
26.8
7.9

AD-239668.1
27.3
3.8

AD-239669.1
27.7
6.0

AD-239670.1
23.1
3.0

AD-239671.1
21.1
7.9

AD-239674.1
16.4
4.9

AD-239676.1
31.3
6.6

AD-239681.1
19.4
5.5

AD-239682.1
39.9
2.9

AD-239684.1
56.3
9.9

AD-239685.1
29.4
6.0

AD-239697.1
26.9
2.2

AD-239713.1
28.4
4.6

AD-239714.1
10.4
1.1

AD-239717.1
18.3
1.5

AD-239721.1
14.4
3.7

AD-239723.1
54.7
4.1

AD-239724.1
18.5
4.7

AD-239725.1
27.2
7.2

AD-239726.1
29.8
4.7

AD-239755.1
14.3
11.2

AD-239757.1
36.0
4.1

AD-239759.1
23.8
5.5

AD-239761.1
73.7
8.8

AD-239764.1
91.7
7.8

AD-239778.1
25.3
8.7

AD-239779.1
20.0
4.1

AD-239780.1
28.9
7.3

AD-239781.1
14.1
3.5

AD-239782.1
22.3
3.8

AD-239783.1
10.1
1.7

AD-239784.1
16.6
5.5

AD-239785.1
15.0
3.4

AD-239786.1
11.9
0.8

AD-239787.1
8.6
2.1

AD-239791.1
15.8
2.3

AD-239793.1
9.9
0.5

AD-239806.1
33.1
11.4

AD-239839.1
40.2
8.9

AD-239840.1
47.9
5.6

AD-239854.1
27.8
9.0

AD-239855.1
16.6
1.4

AD-239876.1
25.6
10.2

AD-239877.1
8.9
0.9

AD-239881.1
11.1
2.7

AD-239905.1
18.2
1.8

AD-239914.1
28.2
4.6

AD-239915.1
37.4
8.6

AD-239941.1
18.0
6.7

AD-239944.1
35.4
5.9

AD-239945.1
18.5
2.6

AD-239946.1
19.5
6.6

AD-239947.1
17.5
4.3

AD-239977.1
13.7
2.7

AD-239979.1
12.5
3.8

AD-239980.1
12.9
2.6

AD-239998.1
32.1
2.1

AD-240019.1
25.1
3.2

AD-240031.1
12.4
2.4

AD-240032.1
30.6
3.2

AD-240033.1
30.8
1.5

AD-240064.1
19.6
4.1

AD-240090.1
96.3
8.5

AD-240091.1
53.6
4.7

AD-240108.1
29.4
1.9

AD-240117.1
85.3
10.6

AD-240124.1
78.2
7.7

AD-240125.1
82.3
17.8

AD-240126.1
56.2
2.0

AD-240135.1
33.4
13.3

AD-240138.1
35.3
4.7

AD-240140.1
12.7
3.5

AD-240142.1
15.9
3.9

AD-240145.1
15.1
1.1

AD-240147.1
24.6
3.7

AD-240148.1
18.9
4.5

AD-240154.1
62.0
4.5

AD-240155.1
24.9
5.6

AD-240156.1
22.8
5.7

AD-240171.1
28.6
5.1

AD-240172.1
36.6
5.3

AD-240192.1
32.6
3.8

AD-240227.1
54.8
10.6

AD-240228.1
51.8
10.9

AD-240237.1
44.2
10.9

AD-240245.1
85.3
14.5

AD-240276.1
27.6
5.2

AD-240278.1
16.7
2.8

AD-240279.1
16.6
7.6

AD-240280.1
13.2
3.3

AD-240308.1
40.6
5.6

AD-240309.1
59.7
8.3

AD-240310.1
43.0
15.0

AD-240311.1
85.9
12.1

AD-240312.1
58.6
8.7

AD-240313.1
32.1
2.0

AD-240315.1
36.0
6.5

AD-240317.1
28.0
1.8

AD-240318.1
18.9
2.2

AD-240319.1
49.2
4.4

AD-240322.1
47.2
7.2

AD-240324.1
31.4
1.6

AD-240326.1
21.3
3.6

AD-240327.1
24.9
5.4

AD-240333.1
12.0
2.4

AD-240364.1
57.4
7.4

AD-240365.1
92.1
2.3

AD-240375.1
97.7
21.7

AD-240378.1
41.5
5.6

AD-240381.1
22.4
7.1

AD-240382.1
22.5
5.9

AD-240383.1
27.0
3.2

AD-240385.1
23.2
4.1

AD-240386.1
29.9
3.4

AD-240387.1
45.7
5.2

AD-240419.1
76.4
17.2

AD-240420.1
50.1
8.4

AD-240421.1
40.9
6.3

AD-240422.1
44.9
11.0

AD-240423.1
30.4
6.5

AD-240431.1
28.7
3.5

AD-240446.1
13.9
1.7

AD-240474.1
41.8
5.0

AD-240475.1
70.9
15.3

AD-240476.1
15.7
3.4

AD-240490.1
27.9
6.3

AD-240494.1
13.8
4.5

AD-240495.1
19.6
0.9

AD-240501.1
47.6
5.9

AD-240502.1
18.7
3.8

AD-240503.1
19.6
7.0

AD-240504.1
17.2
2.6

AD-240505.1
18.3
3.4

AD-240507.1
23.8
3.3

AD-240512.1
24.6
2.4

AD-240513.1
70.6
9.0

AD-240514.1
22.4
3.1

AD-240538.1
40.7
4.2

AD-240540.1
46.7
8.3

AD-240542.1
21.1
4.0

AD-240545.1
26.7
4.4

AD-240546.1
35.2
3.1

AD-240779.1
31.2
4.4

AD-240782.1
27.9
2.8

AD-240787.1
27.4
4.6

AD-240788.1
16.9
3.9

AD-240789.1
32.5
2.9

AD-240790.1
18.9
5.1

AD-240792.1
28.4
9.9

AD-240793.1
22.4
1.9

AD-240794.1
13.9
1.1

AD-240795.1
25.9
5.2

AD-240826.1
15.2
1.9

AD-240830.1
32.1
7.2

AD-240833.1
25.1
5.0

AD-240834.1
15.3
3.1

AD-240837.1
29.2
5.8

AD-240839.1
14.3
2.6

AD-240843.1
8.4
1.7

AD-240844.1
33.8
8.1

AD-240871.1
18.9
3.2

AD-240875.1
18.0
1.6

TABLE 5

HSD17B13 in vitro 50 nM screen in Primary Human Hepatocytes

% of Human Message

Duplex Name
Remaining 50 nM
STDEV 50 nM

AD-238911.1
116.8
40.6

AD-238912.1
83.9
15.4

AD-238913.1
96.3
14.1

AD-238929.1
82.9
14.7

AD-238930.1
86.4
6.6

AD-238932.1
72.4
23.3

AD-238960.1
64.8
28.2

AD-238979.1
48.1
25.1

AD-238991.1
31.3
7.3

AD-239009.1
34.1
9.3

AD-239015.1
90.4
15.8

AD-239016.1
51.7
13.0

AD-239017.1
31.8
10.1

AD-239018.1
75.5
16.0

AD-239019.1
56.0
22.6

AD-239020.1
59.9
19.3

AD-239031.1
46.6
17.3

AD-239062.1
61.9
21.9

AD-239066.1
61.1
17.3

AD-239103.1
41.0
21.7

AD-239110.1
31.8
7.7

AD-239118.1
48.3
3.7

AD-239119.1
56.2
12.7

AD-239131.1
75.2
18.1

AD-239132.1
90.1
34.9

AD-239133.1
52.0
10.7

AD-239146.1
72.1
35.4

AD-239155.1
39.1
9.8

AD-239156.1
79.3
24.4

AD-239177.1
55.1
15.6

AD-239178.1
72.0
21.4

AD-239179.1
57.8
10.5

AD-239180.1
36.3
8.4

AD-239181.1
60.6
19.7

AD-239182.1
42.4
6.4

AD-239183.1
29.3
13.1

AD-239185.1
68.4
21.1

AD-239186.1
87.7
23.4

AD-239187.1
83.2
14.4

AD-239188.1
54.2
13.2

AD-239189.1
74.5
31.3

AD-239190.1
57.4
3.5

AD-239191.1
49.8
21.9

AD-239192.1
33.9
13.5

AD-239193.1
96.3
36.6

AD-239194.1
63.1
18.3

AD-239196.1
43.5
11.6

AD-239198.1
93.8
8.0

AD-239199.1
44.2
21.5

AD-239203.1
68.2
19.0

AD-239214.1
40.3
8.3

AD-239215.1
71.6
25.3

AD-239239.1
53.9
32.4

AD-239240.1
23.8
6.5

AD-239241.1
56.2
10.0

AD-239242.1
46.0
15.0

AD-239243.1
97.6
73.4

AD-239245.1
84.7
9.2

AD-239246.1
37.6
11.2

AD-239247.1
36.5
15.9

AD-239250.1
34.1
6.8

AD-239251.1
19.3
3.7

AD-239253.1
26.3
6.9

AD-239254.1
52.5
13.7

AD-239255.1
52.8
18.1

AD-239290.1
46.3
30.0

AD-239291.1
53.1
25.1

AD-239292.1
49.8
18.6

AD-239293.1
49.5
15.6

AD-239304.1
32.9
10.3

AD-239312.1
83.1
13.5

AD-239313.1
57.4
16.5

AD-239314.1
85.7
13.7

AD-239316.1
50.9
32.4

AD-239317.1
28.0
3.5

AD-239318.1
64.4
26.9

AD-239321.1
34.8
6.9

AD-239332.1
42.0
13.9

AD-239337.1
29.1
11.0

AD-239348.1
22.5
10.5

AD-239362.1
67.8
29.4

AD-239366.1
67.1
26.2

AD-239368.1
41.2
17.8

AD-239372.1
46.4
6.0

AD-239373.1
27.8
4.1

AD-239379.1
19.4
5.7

AD-239380.1
26.5
9.0

AD-239387.1
30.5
14.6

AD-239397.1
18.9
5.4

AD-239411.1
41.8
17.0

AD-239412.1
79.2
22.8

AD-239431.1
39.5
15.3

AD-239433.1
36.9
12.1

AD-239436.1
101.1
17.3

AD-239437.1
37.6
20.8

AD-239438.1
54.0
9.7

AD-239439.1
31.8
6.5

AD-239450.1
67.0
22.6

AD-239451.1
42.4
15.3

AD-239452.1
57.4
3.0

AD-239453.1
45.9
5.9

AD-239475.1
27.2
5.0

AD-239476.1
41.4
16.7

AD-239478.1
38.7
5.7

AD-239479.1
44.0
19.6

AD-239480.1
49.2
10.7

AD-239482.1
31.6
9.4

AD-239483.1
37.5
8.3

AD-239499.1
56.0
12.2

AD-239510.1
51.1
20.1

AD-239520.1
26.5
15.5

AD-239521.1
33.4
19.0

AD-239522.1
36.2
18.2

AD-239534.1
30.7
17.4

AD-239539.1
76.9
31.7

AD-239544.1
21.3
10.9

AD-239566.1
39.1
8.1

AD-239578.1
19.9
12.4

AD-239579.1
24.7
6.7

AD-239611.1
38.8
34.7

AD-239612.1
60.9
15.4

AD-239613.1
76.4
15.6

AD-239617.1
186.1
52.1

AD-239618.1
32.0
5.2

AD-239637.1
22.8
2.8

AD-239641.1
35.9
14.0

AD-239642.1
31.5
28.5

AD-239643.1
12.0
1.7

AD-239645.1
29.6
11.6

AD-239647.1
24.9
7.3

AD-239649.1
35.0
4.2

AD-239650.1
51.2
3.7

AD-239653.1
48.8
26.5

AD-239654.1
105.2
35.2

AD-239662.1
27.5
12.0

AD-239668.1
31.5
9.1

AD-239669.1
42.4
24.3

AD-239670.1
53.3
17.1

AD-239671.1
26.2
11.1

AD-239674.1
17.3
9.5

AD-239676.1
42.1
13.1

AD-239681.1
24.9
11.4

AD-239682.1
12.7
4.5

AD-239684.1
15.2
1.4

AD-239685.1
24.9
6.1

AD-239697.1
15.1
6.2

AD-239713.1
24.6
7.4

AD-239714.1
23.6
18.1

AD-239717.1
36.6
33.7

AD-239721.1
7.3
3.4

AD-239723.1
47.2
12.1

AD-239724.1
29.1
7.8

AD-239725.1
17.7
15.4

AD-239726.1
45.9
13.2

AD-239755.1
19.0
4.6

AD-239757.1
28.6
3.2

AD-239759.1
10.7
3.3

AD-239761.1
60.2
11.2

AD-239764.1
56.4
15.6

AD-239778.1
45.9
32.9

AD-239779.1
18.2
12.0

AD-239780.1
34.5
22.1

AD-239781.1
30.1
16.8

AD-239782.1
37.3
12.4

AD-239783.1
13.0
6.8

AD-239784.1
43.1
14.0

AD-239785.1
9.9
8.9

AD-239786.1
16.9
10.0

AD-239787.1
15.9
9.3

AD-239791.1
11.7
5.4

AD-239793.1
11.1
3.2

AD-239806.1
29.4
13.5

AD-239839.1
60.6
13.8

AD-239840.1
36.7
23.2

AD-239854.1
39.0
20.3

AD-239855.1
31.6
5.2

AD-239876.1
34.6
10.8

AD-239877.1
35.8
21.8

AD-239881.1
47.0
11.9

AD-239905.1
36.6
8.2

AD-239914.1
30.4
13.9

AD-239915.1
51.3
25.7

AD-239941.1
111.8
60.8

AD-239944.1
63.6
23.9

AD-239945.1
58.6
23.0

AD-239946.1
65.3
28.6

AD-239947.1
77.7
16.6

AD-239977.1
54.5
16.5

AD-239979.1
61.4
14.4

AD-239980.1
87.8
41.2

AD-239998.1
54.8
14.7

AD-240019.1
76.7
23.6

AD-240031.1
63.1
18.5

AD-240032.1
51.6
13.5

AD-240033.1
69.7
41.4

AD-240064.1
81.0
32.4

AD-240090.1
145.8
80.6

AD-240091.1
75.2
17.1

AD-240108.1
62.2
18.9

AD-240117.1
54.4
2.3

AD-240124.1
63.3
25.7

AD-240125.1
68.6
18.1

AD-240126.1
77.4
10.9

AD-240135.1
64.9
33.1

AD-240138.1
59.3
17.7

AD-240140.1
65.2
21.2

AD-240142.1
42.9
6.6

AD-240145.1
58.2
10.2

AD-240147.1
56.7
14.3

AD-240148.1
35.0
12.2

AD-240154.1
39.2
19.5

AD-240155.1
57.2
22.1

AD-240156.1
71.9
28.3

AD-240171.1
66.4
22.8

AD-240172.1
50.4
12.9

AD-240192.1
43.5
3.8

AD-240227.1
62.1
19.7

AD-240228.1
50.6
12.7

AD-240237.1
67.2
9.5

AD-240245.1
99.3
21.4

AD-240276.1
49.9
16.6

AD-240278.1
31.8
6.1

AD-240279.1
46.2
2.4

AD-240280.1
68.4
16.4

AD-240308.1
72.7
15.4

AD-240309.1
85.8
2.0

AD-240310.1
58.9
16.1

AD-240311.1
74.2
21.0

AD-240312.1
83.3
7.3

AD-240313.1
70.4
15.1

AD-240315.1
66.1
7.6

AD-240317.1
52.0
11.1

AD-240318.1
65.1
24.1

AD-240319.1
38.8
7.7

AD-240322.1
69.9
10.1

AD-240324.1
79.6
37.5

AD-240326.1
29.8
3.0

AD-240327.1
62.6
11.3

AD-240333.1
60.3
31.5

AD-240364.1
81.0
16.8

AD-240365.1
104.1
31.5

AD-240375.1
111.2
18.0

AD-240378.1
85.1
33.8

AD-240381.1
87.8
18.3

AD-240382.1
51.9
9.5

AD-240383.1
45.6
13.2

AD-240385.1
53.5
6.6

AD-240386.1
63.3
6.8

AD-240387.1
75.8
26.0

AD-240419.1
81.2
11.1

AD-240420.1
69.3
32.6

AD-240421.1
93.8
17.0

AD-240422.1
75.6
38.5

AD-240423.1
75.7
14.1

AD-240431.1
53.2
2.4

AD-240446.1
93.2
30.6

AD-240474.1
74.7
22.8

AD-240475.1
74.4
29.5

AD-240476.1
37.2
2.4

AD-240490.1
82.0
34.8

AD-240494.1
62.3
14.1

AD-240495.1
102.5
39.4

AD-240501.1
33.9
4.9

AD-240502.1
40.6
5.7

AD-240503.1
63.4
31.4

AD-240504.1
55.0
7.7

AD-240505.1
73.0
22.1

AD-240507.1
68.3
23.1

AD-240512.1
55.0
19.6

AD-240513.1
54.9
14.8

AD-240514.1
33.8
16.7

AD-240538.1
51.3
13.3

AD-240540.1
72.1
11.2

AD-240542.1
60.0
9.0

AD-240545.1
45.9
23.8

AD-240546.1
46.2
18.4

AD-240779.1
64.8
22.4

AD-240782.1
77.7
20.3

AD-240787.1
59.6
16.9

AD-240788.1
54.2
12.0

AD-240789.1
58.9
24.6

AD-240790.1
54.0
14.7

AD-240792.1
67.2
9.2

AD-240793.1
54.9
14.6

AD-240794.1
69.3
18.8

AD-240795.1
34.5
7.2

AD-240826.1
56.5
16.8

AD-240830.1
63.7
14.4

AD-240833.1
84.9
28.4

AD-240834.1
50.2
15.1

AD-240837.1
46.3
16.3

AD-240839.1
54.9
18.4

AD-240843.1
46.4
13.9

AD-240844.1
38.2
10.3

AD-240871.1
92.8
35.8

AD-240875.1
72.0
13.2

TABLE 6

HSD17B13 in vitro 50 nM screen in

Primary Cynomolgus Hepatocytes

% of Cyno Message

Duplex Name
Remaining 50 nM
STDEV 50 nM

AD-238911.1
81.2
8.9

AD-238912.1
87.5
6.8

AD-238913.1
62.3
4.1

AD-238929.1
67.1
22.3

AD-238930.1
42.1
4.0

AD-238932.1
54.8
12.2

AD-238960.1
24.6
5.2

AD-238979.1
11.1
7.5

AD-238991.1
30.8
6.2

AD-239009.1
11.4
1.1

AD-239015.1
21.2
3.0

AD-239016.1
7.2
1.4

AD-239017.1
8.9
1.8

AD-239018.1
39.2
4.6

AD-239019.1
22.0
5.5

AD-239020.1
8.8
2.8

AD-239031.1
19.5
7.5

AD-239062.1
20.6
5.6

AD-239066.1
75.5
16.3

AD-239103.1
88.4
33.4

AD-239110.1
11.2
3.0

AD-239118.1
23.1
6.3

AD-239119.1
23.4
4.2

AD-239131.1
98.9
9.0

AD-239132.1
86.0
18.5

AD-239133.1
24.3
8.7

AD-239146.1
11.4
2.8

AD-239155.1
39.4
21.3

AD-239156.1
52.8
11.0

AD-239177.1
23.1
6.7

AD-239178.1
46.3
4.6

AD-239179.1
40.3
8.7

AD-239180.1
21.8
6.2

AD-239181.1
45.5
5.9

AD-239182.1
17.7
3.8

AD-239183.1
10.6
1.5

AD-239185.1
55.9
7.2

AD-239186.1
77.7
13.0

AD-239187.1
61.1
6.8

AD-239188.1
41.0
1.5

AD-239189.1
11.6
2.1

AD-239190.1
48.8
7.9

AD-239191.1
9.0
4.1

AD-239192.1
35.0
12.1

AD-239193.1
68.1
4.9

AD-239194.1
26.7
1.7

AD-239196.1
56.6
10.2

AD-239198.1
80.5
11.0

AD-239199.1
32.8
4.6

AD-239203.1
36.9
6.3

AD-239214.1
15.3
2.7

AD-239215.1
28.9
6.5

AD-239239.1
17.0
2.8

AD-239240.1
15.6
4.4

AD-239241.1
16.8
9.9

AD-239242.1
16.5
5.9

AD-239243.1
13.3
4.2

AD-239245.1
13.7
2.9

AD-239246.1
12.9
8.2

AD-239247.1
18.0
6.5

AD-239250.1
49.8
6.5

AD-239251.1
6.8
0.5

AD-239253.1
11.5
3.8

AD-239254.1
47.4
4.6

AD-239255.1
16.4
7.2

AD-239290.1
75.5
25.9

AD-239291.1
92.3
27.1

AD-239292.1
9.8
2.7

AD-239293.1
46.8
6.3

AD-239304.1
30.7
8.9

AD-239312.1
15.0
3.2

AD-239313.1
8.7
2.4

AD-239314.1
100.1
18.8

AD-239316.1
40.0
9.5

AD-239317.1
12.3
2.0

AD-239318.1
9.3
1.8

AD-239321.1
9.2
2.4

AD-239332.1
48.0
12.4

AD-239337.1
15.4
7.5

AD-239348.1
21.1
6.0

AD-239362.1
75.3
6.7

AD-239366.1
30.7
11.1

AD-239368.1
24.6
6.2

AD-239372.1
45.5
2.7

AD-239373.1
33.3
7.5

AD-239379.1
8.1
2.6

AD-239380.1
7.2
1.1

AD-239387.1
36.2
5.3

AD-239397.1
12.3
3.4

AD-239411.1
31.6
10.7

AD-239412.1
59.5
7.0

AD-239431.1
13.2
7.9

AD-239433.1
33.0
10.1

AD-239436.1
71.5
14.7

AD-239437.1
10.4
2.6

AD-239438.1
12.3
2.7

AD-239439.1
79.2
11.8

AD-239450.1
61.3
2.2

AD-239451.1
29.7
8.7

AD-239452.1
78.7
17.3

AD-239453.1
38.5
10.6

AD-239475.1
11.4
2.2

AD-239476.1
39.5
6.9

AD-239478.1
59.3
9.4

AD-239479.1
14.7
3.6

AD-239480.1
31.6
6.6

AD-239482.1
13.7
3.3

AD-239483.1
14.1
3.8

AD-239499.1
111.5
13.7

AD-239510.1
16.0
4.0

AD-239520.1
11.3
5.2

AD-239521.1
19.8
3.7

AD-239522.1
12.5
3.8

AD-239534.1
31.1
8.4

AD-239539.1
9.3
3.5

AD-239544.1
24.0
3.2

AD-239566.1
11.9
7.0

AD-239578.1
25.7
5.5

AD-239579.1
28.1
5.9

AD-239611.1
37.4
7.8

AD-239612.1
36.2
8.4

AD-239613.1
13.5
3.3

AD-239617.1
46.2
9.3

AD-239618.1
15.9
1.2

AD-239637.1
33.6
13.4

AD-239641.1
42.3
7.4

AD-239642.1
34.4
5.7

AD-239643.1
9.7
2.0

AD-239645.1
16.1
3.4

AD-239647.1
19.0
4.0

AD-239649.1
70.4
6.0

AD-239650.1
65.8
12.5

AD-239653.1
16.4
3.6

AD-239654.1
18.3
6.5

AD-239662.1
8.4
4.1

AD-239668.1
13.2
4.2

AD-239669.1
53.6
14.0

AD-239670.1
14.4
3.6

AD-239671.1
12.1
2.9

AD-239674.1
14.8
4.0

AD-239676.1
20.9
4.8

AD-239681.1
9.4
0.6

AD-239682.1
8.9
1.3

AD-239684.1
12.0
2.9

AD-239685.1
10.8
3.7

AD-239697.1
11.8
3.3

AD-239713.1
11.4
1.8

AD-239714.1
7.8
2.8

AD-239717.1
10.4
1.5

AD-239721.1
27.2
6.4

AD-239723.1
81.2
14.9

AD-239724.1
11.0
3.7

AD-239725.1
24.1
3.0

AD-239726.1
27.3
7.7

AD-239755.1
7.8
3.3

AD-239757.1
30.3
3.4

AD-239759.1
6.7
1.7

AD-239761.1
49.7
11.8

AD-239764.1
53.5
11.4

AD-239778.1
26.8
7.8

AD-239779.1
63.3
20.5

AD-239780.1
85.5
17.8

AD-239781.1
26.4
6.0

AD-239782.1
22.6
6.7

AD-239783.1
9.7
1.9

AD-239784.1
20.2
3.8

AD-239785.1
23.6
6.7

AD-239786.1
38.3
6.1

AD-239787.1
27.6
2.5

AD-239791.1
17.8
2.1

AD-239793.1
6.8
1.2

AD-239806.1
27.8
2.9

AD-239839.1
11.9
5.6

AD-239840.1
10.3
1.8

AD-239854.1
58.6
8.9

AD-239855.1
23.3
9.3

AD-239876.1
42.8
20.3

AD-239877.1
30.6
10.4

AD-239881.1
53.4
5.5

AD-239905.1
50.1
17.6

AD-239914.1
37.3
5.2

AD-239915.1
34.8
5.7

AD-239941.1
66.8
14.1

AD-239944.1
117.7
12.5

AD-239945.1
23.2
2.7

AD-239946.1
14.0
5.9

AD-239947.1
25.8
1.6

AD-239977.1
27.3
4.4

AD-239979.1
25.5
4.8

AD-239980.1
35.5
6.4

AD-239998.1
32.7
4.0

AD-240019.1
17.9
2.6

AD-240031.1
15.3
1.9

AD-240032.1
23.4
2.4

AD-240033.1
22.2
3.7

AD-240064.1
47.1
5.2

AD-240090.1
25.7
5.9

AD-240091.1
31.4
8.2

AD-240108.1
118.3
27.0

AD-240117.1
56.6
5.6

AD-240124.1
43.5
8.1

AD-240125.1
54.0
6.8

AD-240126.1
30.7
7.0

AD-240135.1
14.7
0.8

AD-240138.1
36.6
13.8

AD-240140.1
33.6
10.9

AD-240142.1
19.4
4.7

AD-240145.1
27.6
9.1

AD-240147.1
18.1
3.4

AD-240148.1
9.0
1.7

AD-240154.1
23.0
1.0

AD-240155.1
18.2
1.7

AD-240156.1
15.1
2.2

AD-240171.1
29.9
6.9

AD-240172.1
31.9
8.0

AD-240192.1
39.7
5.7

AD-240227.1
71.4
15.6

AD-240228.1
35.8
22.3

AD-240237.1
43.3
17.3

AD-240245.1
41.5
5.1

AD-240276.1
20.6
4.4

AD-240278.1
11.6
1.8

AD-240279.1
20.1
0.5

AD-240280.1
51.0
9.5

AD-240308.1
80.5
19.3

AD-240309.1
63.5
10.3

AD-240310.1
45.2
10.3

AD-240311.1
42.6
14.8

AD-240312.1
22.3
1.3

AD-240313.1
21.6
4.5

AD-240315.1
26.4
8.2

AD-240317.1
17.8
3.6

AD-240318.1
20.5
2.5

AD-240319.1
23.5
6.5

AD-240322.1
37.5
2.7

AD-240324.1
37.1
13.9

AD-240326.1
11.2
3.4

AD-240327.1
28.7
4.0

AD-240333.1
32.0
5.8

AD-240364.1
34.1
13.5

AD-240365.1
58.8
18.7

AD-240375.1
86.7
17.8

AD-240378.1
49.8
14.8

AD-240381.1
56.0
12.0

AD-240382.1
35.7
7.9

AD-240383.1
27.5
5.5

AD-240385.1
15.8
2.0

AD-240386.1
18.7
2.0

AD-240387.1
33.3
13.0

AD-240419.1
32.8
4.2

AD-240420.1
31.5
4.5

AD-240421.1
21.5
4.7

AD-240422.1
19.9
4.6

AD-240423.1
13.1
2.3

AD-240431.1
16.1
3.3

AD-240446.1
16.6
3.5

AD-240474.1
28.7
10.8

AD-240475.1
32.7
11.9

AD-240476.1
11.0
1.8

AD-240490.1
38.5
5.6

AD-240494.1
37.3
14.0

AD-240495.1
17.4
4.6

AD-240501.1
29.5
6.7

AD-240502.1
16.3
1.2

AD-240503.1
12.4
2.5

AD-240504.1
15.6
4.4

AD-240505.1
44.8
15.3

AD-240507.1
47.0
12.7

AD-240512.1
31.9
3.9

AD-240513.1
44.2
3.8

AD-240514.1
13.0
2.4

AD-240538.1
35.5
6.4

AD-240540.1
40.0
12.4

AD-240542.1
65.6
15.4

AD-240545.1
18.7
2.3

AD-240546.1
32.4
5.0

AD-240779.1
33.8
8.0

AD-240782.1
33.6
7.3

AD-240787.1
35.4
3.4

AD-240788.1
12.5
1.3

AD-240789.1
42.6
8.0

AD-240790.1
21.9
2.1

AD-240792.1
22.0
2.8

AD-240793.1
22.7
3.7

AD-240794.1
37.5
9.5

AD-240795.1
18.9
1.9

AD-240826.1
38.9
6.9

AD-240830.1
32.9
2.4

AD-240833.1
41.6
9.4

AD-240834.1
17.2
3.6

AD-240837.1
42.1
7.9

AD-240839.1
18.4
3.7

AD-240843.1
17.1
5.9

AD-240844.1
19.8
3.8

AD-240871.1
16.2
2.1

AD-240875.1
48.1
27.7

Example 2. iRNA Design, Synthesis, and Selection

This Example describes methods for the design, synthesis, and selection of additional HSD17B13 iRNA agents.

Bioinformatics

In Vitro Cos-7 (Dual-Luciferase psiCHECK2 Human HSD17B13 Vector) and Primary Cynomolgus Hepatocytes Screening

Dual-Glo® Luciferase Assay

Cos-7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Multi-dose experiments were performed at 10 nM and 0.1 nM. siRNA and psiCHECK2-HSD17B13 (NM_178135 cloned into XhoI-NotI sites) plasmid transfection was carried out by adding 5 μl of siRNA duplexes and 5 μl (5 ng) of psiCHECK2-HSD17B13 plasmid per well along with 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine 2000 per well (Invitrogen, Carlsbad Calif. cat #13778-150) and then incubated at room temperature for 15 minutes. The mixture was then added to the cells which were re-suspended in 35 μl of fresh complete media. The transfected cells were incubated at 37° C. in an atmosphere of 5% CO₂.

Forty-eight hours after the siRNAs and psiCHECK2-HSD17B13 plasmid were transfected; Firefly (transfection control) and Renilla (fused to HSD17B13 target sequence) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 20 μl of Dual-Glo® Luciferase Reagent equal to the culture medium volume to each well and mixing. The mixture was incubated at room temperature for 30 minutes before luminescense (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. 20 μl of room temperature Dual-Glo® Stop & Glo® Reagent was added to each well and the plates were incubated for 10-15 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenched the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (HSD17B13) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-targeting siRNA. All transfections were done at n=2 or greater.

Cell Culture and Transfections

Primary Cynomolgus Hepatocytes (Bioreclamation/IVT) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of RNAiMAX per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well, with 4 replicates of each SiRNA duplex, into a 384-well plate, and incubating the plate at room temperature for 15 minutes. 40 μl of InVitroGRO CP plating media (Bioreclamation/IVT) containing ˜5×10³cells were then added to the siRNA mixture. Cells were incubated for 48 hours prior to RNA purification. Multi-dose experiments were performed at 10 nM and 0.1 nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 70 μl of Lysis/Binding Buffer and 10 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150 μl Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured and supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

10 μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H₂O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 h at 37° C.

Real Time PCR

2 μl of cDNA were added to a master mix containing 0.5 μl of Cyno GAPDH TaqMan Probe (forward primer: 5′-GCATCCTGGGCTACACTGA-3′ (SEQ ID NO: 4483), reverse primer: 5′-TGGGTGTCGCTGTTGAAGTC-3′ (SEQ ID NO: 4484), probe: 5′HEX-CCAGGTGGTCTCCTCC-3′BHQ-1 (SEQ ID NO: 4485)), and 0.5 μl HSD17B13 cynomolgus probe (Mf02888851_m1) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384-well plate (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). Each duplex was tested at least two times and data were normalized to cells transfected with a non-targeting control siRNA. To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with a non-targeting control siRNA.

Results

Table 7 provides HSD17B13 ELF7 GNA7 modified sequences. Table 8 provides HSD17B13 ELF7 GNA7 unmodified sequences. Results from the HSD17B13 ELF7 GNA7 modified sequences in vitro 10 nM screen in Cos-7 and primary cynomolgus hepatocytes are summarized in Table 9.

Similarly, Table 10 provides HSD17B13 NonF DNA modified sequences. Table 11 provides HSD17B13 NonF DNA unmodified sequences. Shown in Table 12 are results from the HSD17B13 NonF DNA modified sequences in vitro 10 nM screen in Cos-7 and primary cynomolgus hepatocytes. “NonF” indicates that these RNAi agents lack fluoro modifications.

TABLE 7

HSD17B13 ELF7 GNA7 Modified Sequence*

SEQ

SEQ

SEQ

ID

ID
mRNA target
ID

Duplex Name
Sense Sequence 5′ to 3′
NO:
Antisense Sequence 5′ to 3′
NO:
sequence 5′ to 3′
NO:

AD-286958.1
asgsuaccUfcCfUfCfccuagg
2981
usAfsgucc(Tgn)agggagGfaGfgu
302
ACAGUACCUCCUCCCUAG
602

acuaL96

acusgsu

GACUA

AD-287001.1
uscscuucUfgCfUfUfcugauc
2982
usGfsguga(Tgn)cagaagCfaGfaa
303
AAUCCUUCUGCUUCUGAU
603

accaL96

ggasusu

CACCA

AD-287012.1
csasucauCfuAfCfUfccuacu
2983
usCfscaag(Tgn)aggaguAfgAfug
304
ACCAUCAUCUACUCCUAC
604

uggaL96

augsgsu

UUGGA

AD-287014.1
uscsuacuCfcUfAfCfuuggag
2984
asCfsgacu(Cgn)caaguaGfgAfgu
305
CAUCUACUCCUACUUGGA
605

ucguL96

agasusg

GUCGU

AD-287015.1
csusacucCfuAfCfUfuggagu
2985
asAfscgac(Tgn)ccaaguAfgGfag
306
AUCUACUCCUACUUGGAG
606

cguuL96

uagsasu

UCGUU

AD-287017.1
gsusaccuCfcUfCfCfcuagga
2986
asUfsaguc(Cgn)uagggaGfgAfgg
307
CAGUACCUCCUCCCUAGG
607

cuauL96

uacsusg

ACUAC

AD-287026.1
ascsacaaGfgAfCfUfgaacca
2987
asUfsucug(Ggn)uucaguCfcUfug
308
CUACACAAGGACUGAACC
608

gaauL96

ugusasg

AGAAG

AD-287059.1
ascscaucAfuCfUfAfcuccua
2988
asAfsagua(Ggn)gaguagAfuGfau
309
UCACCAUCAUCUACUCCU
609

cuuuL96

ggusgsa

ACUUG

AD-287060.1
cscsaucaUfcUfAfCfuccuac
2989
asCfsaagu(Agn)ggaguaGfaUfga
310
CACCAUCAUCUACUCCUA
610

uuguL96

uggsusg

CUUGG

AD-287061.1
asuscaucUfaCfUfCfcuacuu
2990
asUfsccaa(Ggn)uaggagUfaGfau
311
CCAUCAUCUACUCCUACU
611

ggauL96

gausgsg

UGGAG

AD-287062.1
csasucuaCfuCfCfUfacuugg
2991
asAfscucc(Agn)aguaggAfgUfag
312
AUCAUCUACUCCUACUUG
612

aguuL96

augsasu

GAGUC

AD-287064.1
usascuccUfaCfUfUfggaguc
2992
asAfsacga(Cgn)uccaagUfaGfga
313
UCUACUCCUACUUGGAGU
613

guuuL96

guasgsa

CGUUG

AD-287065.1
ascsuccuAfcUfUfGfgagucg
2993
asCfsaacg(Agn)cuccaaGfuAfgg
314
CUACUCCUACUUGGAGUC
614

uuguL96

agusasg

GUUGG

AD-287066.1
csusccuaCfuUfGfGfagucgu
2994
asCfscaac(Ggn)acuccaAfgUfag
315
UACUCCUACUUGGAGUCG
615

ugguL96

gagsusa

UUGGU

AD-287126.1
uscscuacUfuGfGfAfgucguu
2995
asAfsccaa(Cgn)gacuccAfaGfua
316
ACUCCUACUUGGAGUCGU
616

gguuL96

ggasgsu

UGGUG

AD-287200.1
asascugcAfgCfUfGfagugcc
2996
usUfsucgg(Cgn)acucagCfuGfca
317
GAAACUGCAGCUGAGUGC
617

gaaaL96

guususc

CGAAA

AD-287201.1
ascsugcaGfcUfGfAfgugccg
2997
usUfsuucg(Ggn)cacucaGfcUfgc
318
AAACUGCAGCUGAGUGCC
618

aaaaL96

agususu

GAAAA

AD-287202.1
usgscagcUfgAfGfUfgccgaa
2998
asGfsuuuu(Cgn)ggcacuCfaGfcu
319
ACUGCAGCUGAGUGCCGA
619

aacuL96

gcasgsu

AAACU

AD-287203.1
gscsagcuGfaGfUfGfccgaaa
2999
usAfsguuu(Tgn)cggcacUfcAfgc
320
CUGCAGCUGAGUGCCGAA
620

acuaL96

ugcsasg

AACUA

AD-287205.1
asgsugccGfaAfAfAfcuaggc
3000
usGfsacgc(Cgn)uaguuuUfcGfgc
321
UGAGUGCCGAAAACUAGG
621

gucaL96

acuscsa

CGUCA

AD-287206.1
usgsccgaAfaAfCfUfaggcgu
3001
asGfsugac(Ggn)ccuaguUfuUfcg
322
AGUGCCGAAAACUAGGCG
622

cacuL96

gcascsu

UCACU

AD-287227.1
csusguggGfaUfAfUfuaauaa
3002
asCfsgcuu(Agn)uuaauaUfcCfca
323
UUCUGUGGGAUAUUAAUA
623

gcguL96

cagsasa

AGCGC

AD-287246.1
csusgcagCfuGfAfGfugccga
24
asUfsuuuc(Ggn)gcacucAfgCfug
324
AACUGCAGCUGAGUGCCG
624

aaauL96

cagsusu

AAAAC

AD-287247.1
csasgcugAfgUfGfCfcgaaaa
25
asUfsaguu(Tgn)ucggcaCfuCfag
325
UGCAGCUGAGUGCCGAAA
625

cuauL96

cugscsa

ACUAG

AD-287248.1
asgscugaGfuGfCfCfgaaaac
26
asCfsuagu(Tgn)uucggcAfcUfca
326
GCAGCUGAGUGCCGAAAA
626

uaguL96

gcusgsc

CUAGG

AD-287249.1
gscsugagUfgCfCfGfaaaacu
27
asCfscuag(Tgn)uuucggCfaCfuc
327
CAGCUGAGUGCCGAAAAC
627

agguL96

agcsusg

UAGGC

AD-287250.1
csusgaguGfcCfGfAfaaacua
28
asGfsccua(Ggn)uuuucgGfcAfcu
328
AGCUGAGUGCCGAAAACU
628

ggcuL96

cagscsu

AGGCG

AD-287251.1
gsasgugcCfgAfAfAfacuagg
29
asAfscgcc(Tgn)aguuuuCfgGfca
329
CUGAGUGCCGAAAACUAG
629

cguuL96

cucsasg

GCGUC

AD-287252.1
gsusgccgAfaAfAfCfuaggcg
30
asUfsgacg(Cgn)cuaguuUfuCfgg
330
GAGUGCCGAAAACUAGGC
630

ucauL96

cacsusc

GUCAC

AD-287253.1
gscscgaaAfaCfUfAfggcguc
31
asAfsguga(Cgn)gccuagUfuUfuc
331
GUGCCGAAAACUAGGCGU
631

acuuL96

ggcsasc

CACUG

AD-287254.1
cscsgaaaAfcUfAfGfgcguca
32
asCfsagug(Agn)cgccuaGfuUfuu
332
UGCCGAAAACUAGGCGUC
632

cuguL96

cggscsa

ACUGC

AD-287258.1
ascsuaggCfgUfCfAfcugcgc
33
asCfsaugc(Ggn)cagugaCfgCfcu
333
AAACUAGGCGUCACUGCG
633

auguL96

agususu

CAUGC

AD-287287.1
gsasucuaUfcGfCfUfcucuaa
34
usGfsauuu(Agn)gagagcGfaUfag
334
GAGAUCUAUCGCUCUCUA
634

aucaL96

aucsusc

AAUCA

AD-287288.1
csusaucgCfuCfUfCfuaaauc
35
asCfscuga(Tgn)uuagagAfgCfga
335
AUCUAUCGCUCUCUAAAU
635

agguL96

uagsasu

CAGGU

AD-287289.1
asuscgcuCfuCfUfAfaaucag
36
usCfsaccu(Ggn)auuuagAfgAfgc
336
CUAUCGCUCUCUAAAUCA
636

gugaL96

gausasg

GGUGA

AD-287290.1
uscsgcucUfcUfAfAfaucagg
37
usUfscacc(Tgn)gauuuaGfaGfag
337
UAUCGCUCUCUAAAUCAG
637

ugaaL96

cgasusa

GUGAA

AD-287293.1
uscsucuaAfaUfCfAfggugaa
38
usUfsucuu(Cgn)accugaUfuUfag
338
GCUCUCUAAAUCAGGUGA
638

gaaaL96

agasgsc

AGAAA

AD-287316.1
gsusggugAfaUfAfAfugcugg
39
usGfsuccc(Agn)gcauuaUfuCfac
339
UCGUGGUGAAUAAUGCUG
639

gacaL96

cacsgsa

GGACA

AD-287320.1
gsasauaaUfgCfUfGfggacag
40
usAfsuacu(Ggn)ucccagCfaUfua
340
GUGAAUAAUGCUGGGACA
640

uauaL96

uucsasc

GUAUA

AD-287321.1
asasuaauGfcUfGfGfgacagu
41
asUfsauac(Tgn)gucccaGfcAfuu
341
UGAAUAAUGCUGGGACAG
641

auauL96

auuscsa

UAUAU

AD-287324.1
gsgsgacaGfuAfUfAfuccagc
42
asUfscggc(Tgn)ggauauAfcUfgu
342
CUGGGACAGUAUAUCCAG
642

cgauL96

cccsasg

CCGAU

AD-287325.1
gsascaguAfuAfUfCfcagccg
43
asGfsaucg(Ggn)cuggauAfuAfcu
343
GGGACAGUAUAUCCAGCC
643

aucuL96

gucscsc

GAUCU

AD-287326.1
ascsaguaUfaUfCfCfagccga
44
asAfsgauc(Ggn)gcuggaUfaUfac
344
GGACAGUAUAUCCAGCCG
644

ucuuL96

uguscsc

AUCUU

AD-287327.1
asgsuauaUfcCfAfGfccgauc
45
asGfsaaga(Tgn)cggcugGfaUfau
345
ACAGUAUAUCCAGCCGAU
645

uucuL96

acusgsu

CUUCU

AD-287344.1
asuscuauCfgCfUfCfucuaaa
46
asUfsgauu(Tgn)agagagCfgAfua
346
AGAUCUAUCGCUCUCUAA
646

ucauL96

gauscsu

AUCAG

AD-287346.1
usasucgcUfcUfCfUfaaauca
47
asAfsccug(Agn)uuuagaGfaGfcg
347
UCUAUCGCUCUCUAAAUC
647

gguuL96

auasgsa

AGGUG

AD-287347.1
csgscucuCfuAfAfAfucaggu
48
asUfsucac(Cgn)ugauuuAfgAfga
348
AUCGCUCUCUAAAUCAGG
648

gaauL96

gcgsasu

UGAAG

AD-287366.1
csgsugguGfaAfUfAfaugcug
49
asUfsccca(Ggn)cauuauUfcAfcc
349
AUCGUGGUGAAUAAUGCU
649

ggauL96

acgsasu

GGGAC

AD-287367.1
usgsgugaAfuAfAfUfgcuggg
50
asUfsgucc(Cgn)agcauuAfuUfca
350
CGUGGUGAAUAAUGCUGG
650

acauL96

ccascsg

GACAG

AD-287370.1
asusgcugGfgAfCfAfguauau
51
asUfsggau(Agn)uacuguCfcCfag
351
UAAUGCUGGGACAGUAUA
651

ccauL96

caususa

UCCAG

AD-287371.1
usgscuggGfaCfAfGfuauauc
52
asCfsugga(Tgn)auacugUfcCfca
352
AAUGCUGGGACAGUAUAU
652

caguL96

gcasusu

CCAGC

AD-287372.1
gscsugggAfcAfGfUfauaucc
53
asGfscugg(Agn)uauacuGfuCfcc
353
AUGCUGGGACAGUAUAUC
653

agcuL96

agcsasu

CAGCC

AD-287373.1
csusgggaCfaGfUfAfuaucca
54
asGfsgcug(Ggn)auauacUfgUfcc
354
UGCUGGGACAGUAUAUCC
654

gccuL96

cagscsa

AGCCG

AD-287374.1
gsgsacagUfaUfAfUfccagcc
55
asAfsucgg(Cgn)uggauaUfaCfug
355
UGGGACAGUAUAUCCAGC
655

gauuL96

uccscsa

CGAUC

AD-287375.1
csasguauAfuCfCfAfgccgau
56
asAfsagau(Cgn)ggcuggAfuAfua
356
GACAGUAUAUCCAGCCGA
656

cuuuL96

cugsusc

UCUUC

AD-287391.1
cscsaaggAfuGfAfAfgagauu
57
usGfsguaa(Tgn)cucuucAfuCfcu
357
CACCAAGGAUGAAGAGAU
657

accaL96

uggsusg

UACCA

AD-287392.1
csasaggaUfgAfAfGfagauua
58
usUfsggua(Agn)ucucuuCfaUfcc
358
ACCAAGGAUGAAGAGAUU
658

ccaaL96

uugsgsu

ACCAA

AD-287406.1
ususugagGfuCfAfAfcauccu
59
usCfscuag(Ggn)auguugAfcCfuc
359
CAUUUGAGGUCAACAUCC
659

aggaL96

aaasusg

UAGGA

AD-287407.1
usgsagguCfaAfCfAfuccuag
60
usGfsuccu(Agn)ggauguUfgAfcc
360
UUUGAGGUCAACAUCCUA
660

gacaL96

ucasasa

GGACA

AD-287408.1
gsasggucAfaCfAfUfccuagg
61
asUfsgucc(Tgn)aggaugUfuGfac
361
UUGAGGUCAACAUCCUAG
661

acauL96

cucsasa

GACAU

AD-287410.1
gsgsucaaCfaUfCfCfuaggac
62
asAfsaugu(Cgn)cuaggaUfgUfug
362
GAGGUCAACAUCCUAGGA
662

auuuL96

accsusc

CAUUU

AD-287411.1
gsuscaacAfuCfCfUfaggaca
63
asAfsaaug(Tgn)ccuaggAfuGfuu
363
AGGUCAACAUCCUAGGAC
663

uuuuL96

gacscsu

AUUUU

AD-287413.1
ascsauccUfaGfGfAfcauuuu
64
usCfscaaa(Agn)auguccUfaGfga
364
CAACAUCCUAGGACAUUU
664

uggaL96

ugususg

UUGGA

AD-287414.1
csasuccuAfgGfAfCfauuuuu
65
asUfsccaa(Agn)aaugucCfuAfgg
365
AACAUCCUAGGACAUUUU
665

ggauL96

augsusu

UGGAU

AD-287415.1
uscscuagGfaCfAfUfuuuugg
66
usGfsaucc(Agn)aaaaugUfcCfua
366
CAUCCUAGGACAUUUUUG
666

aucaL96

ggasusg

GAUCA

AD-287418.1
asgsgacaUfuUfUfUfggauca
67
usUfsugug(Agn)uccaaaAfaUfgu
367
CUAGGACAUUUUUGGAUC
667

caaaL96

ccusasg

ACAAA

AD-287419.1
gsgsacauUfuUfUfGfgaucac
68
usUfsuugu(Ggn)auccaaAfaAfug
368
UAGGACAUUUUUGGAUCA
668

aaaaL96

uccsusa

CAAAA

AD-287421.1
ususuuugGfaUfCfAfcaaaag
69
asGfsugcu(Tgn)uugugaUfcCfaa
369
CAUUUUUGGAUCACAAAA
669

cacuL96

aaasusg

GCACU

AD-287424.1
usgsgaucAfcAfAfAfagcacu
70
asAfsgaag(Tgn)gcuuuuGfuGfau
370
UUUGGAUCACAAAAGCAC
670

ucuuL96

ccasasa

UUCUU

AD-287427.1
csasaaagCfaCfUfUfcuucca
71
usCfsgaug(Ggn)aagaagUfgCfuu
371
CACAAAAGCACUUCUUCC
671

ucgaL96

uugsusg

AUCGA

AD-287428.1
asasaagcAfcUfUfCfuuccau
72
asUfscgau(Ggn)gaagaaGfuGfcu
372
ACAAAAGCACUUCUUCCA
672

cgauL96

uuusgsu

UCGAU

AD-287429.1
asasgcacUfuCfUfUfccaucg
73
usCfsaucg(Agn)uggaagAfaGfug
373
AAAAGCACUUCUUCCAUC
673

augaL96

cuususu

GAUGA

AD-287430.1
asgscacuUfcUfUfCfcaucga
74
asUfscauc(Ggn)auggaaGfaAfgu
374
AAAGCACUUCUUCCAUCG
674

ugauL96

gcususu

AUGAU

AD-287441.1
gsgsagagAfaAfUfCfauggcc
75
asUfsgugg(Cgn)caugauUfuCfuc
375
AUGGAGAGAAAUCAUGGC
675

acauL96

uccsasu

CACAU

AD-287453.1
ascscaagGfaUfGfAfagagau
76
asGfsuaau(Cgn)ucuucaUfcCfuu
376
GCACCAAGGAUGAAGAGA
676

uacuL96

ggusgsc

UUACC

AD-287462.1
asgsacauUfuGfAfGfgucaac
77
asGfsaugu(Tgn)gaccucAfaAfug
377
CAAGACAUUUGAGGUCAA
677

aucuL96

ucususg

CAUCC

AD-287463.1
csasuuugAfgGfUfCfaacauc
78
asUfsagga(Tgn)guugacCfuCfaa
378
GACAUUUGAGGUCAACAU
678

cuauL96

augsusc

CCUAG

AD-287464.1
asusuugaGfgUfCfAfacaucc
79
asCfsuagg(Agn)uguugaCfcUfca
379
ACAUUUGAGGUCAACAUC
679

uaguL96

aausgsu

CUAGG

AD-287465.1
ususgaggUfcAfAfCfauccua
80
asUfsccua(Ggn)gauguuGfaCfcu
380
AUUUGAGGUCAACAUCCU
680

ggauL96

caasasu

AGGAC

AD-287466.1
csasacauCfcUfAfGfgacauu
81
csAfsaaaa(Tgn)guccuaGfgAfug
381
GUCAACAUCCUAGGACAU
681

uuugL96

uugsasc

UUUUG

AD-287467.1
asascaucCfuAfGfGfacauuu
82
asCfsaaaa(Agn)uguccuAfgGfau
382
UCAACAUCCUAGGACAUU
682

uuguL96

guusgsa

UUUGG

AD-287468.1
asusccuaGfgAfCfAfuuuuug
83
asAfsucca(Agn)aaauguCfcUfag
383
ACAUCCUAGGACAUUUUU
683

gauuL96

gausgsu

GGAUC

AD-287469.1
cscsuaggAfcAfUfUfuuugga
84
asUfsgauc(Cgn)aaaaauGfuCfcu
384
AUCCUAGGACAUUUUUGG
684

ucauL96

aggsasu

AUCAC

AD-287470.1
gsascauuUfuUfGfGfaucaca
85
asUfsuuug(Tgn)gauccaAfaAfau
385
AGGACAUUUUUGGAUCAC
685

aaauL96

gucscsu

AAAAG

AD-287474.1
gsgsaucaCfaAfAfAfgcacuu
86
asAfsagaa(Ggn)ugcuuuUfgUfga
386
UUGGAUCACAAAAGCACU
686

cuuuL96

uccsasa

UCUUC

AD-287476.1
csascaaaAfgCfAfCfuucuuc
87
asAfsugga(Agn)gaagugCfuUfuu
387
AUCACAAAAGCACUUCUU
687

cauuL96

gugsasu

CCAUC

AD-287477.1
ascsaaaaGfcAfCfUfucuucc
88
asGfsaugg(Agn)agaaguGfcUfuu
388
UCACAAAAGCACUUCUUC
688

aucuL96

ugusgsa

CAUCG

AD-287478.1
asasagcaCfuUfCfUfuccauc
89
asAfsucga(Tgn)ggaagaAfgUfgc
389
CAAAAGCACUUCUUCCAU
689

gauuL96

uuususg

CGAUG

AD-287479.1
gscsacuuCfuUfCfCfaucgau
90
asAfsucau(Cgn)gauggaAfgAfag
390
AAGCACUUCUUCCAUCGA
690

gauuL96

ugcsusu

UGAUG

AD-287491.1
asgsaaauCfaUfGfGfccacau
91
asAfscgau(Ggn)uggccaUfgAfuu
391
AGAGAAAUCAUGGCCACA
691

cguuL96

ucuscsu

UCGUC

AD-287492.1
asasaucaUfgGfCfCfacaucg
92
asUfsgacg(Agn)uguggcCfaUfga
392
AGAAAUCAUGGCCACAUC
692

ucauL96

uuuscsu

GUCAC

AD-287517.1
ususccuuAfcCfUfCfauccca
93
asAfsuaug(Ggn)gaugagGfuAfag
393
GAUUCCUUACCUCAUCCC
693

uauuL96

gaasusc

AUAUU

AD-287520.1
ascscucaUfcCfCfAfuauugu
94
usGfsgaac(Agn)auauggGfaUfga
394
UUACCUCAUCCCAUAUUG
694

uccaL96

ggusasa

UUCCA

AD-287521.1
uscsauccCfaUfAfUfuguucc
95
usGfscugg(Agn)acaauaUfgGfga
395
CCUCAUCCCAUAUUGUUC
695

agcaL96

ugasgsg

CAGCA

AD-287522.1
csasucccAfuAfUfUfguucca
96
usUfsgcug(Ggn)aacaauAfuGfgg
396
CUCAUCCCAUAUUGUUCC
696

gcaaL96

augsasg

AGCAA

AD-287524.1
uscsccauAfuUfGfUfuccagc
97
asUfsuugc(Tgn)ggaacaAfuAfug
397
CAUCCCAUAUUGUUCCAG
697

aaauL96

ggasusg

CAAAU

AD-287525.1
cscscauaUfuGfUfUfccagca
98
asAfsuuug(Cgn)uggaacAfaUfau
398
AUCCCAUAUUGUUCCAGC
698

aauuL96

gggsasu

AAAUU

AD-287537.1
usgsuuggCfuUfUfCfacagag
99
asGfsaccu(Cgn)ugugaaAfgCfca
399
GCUGUUGGCUUUCACAGA
699

gucuL96

acasgsc

GGUCU

AD-287539.1
gsgscuuuCfaCfAfGfaggucu
100
usGfsucag(Agn)ccucugUfgAfaa
400
UUGGCUUUCACAGAGGUC
700

gacaL96

gccsasa

UGACA

AD-287540.1
gscsuuucAfcAfGfAfggucug
101
asUfsguca(Ggn)accucuGfuGfaa
401
UGGCUUUCACAGAGGUCU
701

acauL96

agcscsa

GACAU

AD-287543.1
csascagaGfgUfCfUfgacauc
102
usUfscuga(Tgn)gucagaCfcUfcu
402
UUCACAGAGGUCUGACAU
702

agaaL96

gugsasa

CAGAA

AD-287546.1
asgsgucuGfaCfAfUfcagaac
103
usGfsaagu(Tgn)cugaugUfcAfga
403
AGAGGUCUGACAUCAGAA
703

uucaL96

ccuscsu

CUUCA

AD-287568.1
uscscuuaCfcUfCfAfucccau
104
asAfsauau(Ggn)ggaugaGfgUfaa
404
AUUCCUUACCUCAUCCCA
704

auuuL96

ggasasu

UAUUG

AD-287591.1
ususcacaGfaGfGfUfcugaca
105
asUfsgaug(Tgn)cagaccUfcUfgu
405
CUUUCACAGAGGUCUGAC
705

ucauL96

gaasasg

AUCAG

AD-287610.1
ususgggaAfaAfAfCfugguau
106
usUfsugau(Agn)ccaguuUfuUfcc
406
CCUUGGGAAAAACUGGUA
706

caaaL96

caasgsg

UCAAA

AD-287615.1
ascsugguAfuCfAfAfaaccuc
107
asCfsauga(Ggn)guuuugAfuAfcc
407
AAACUGGUAUCAAAACCU
707

auguL96

agususu

CAUGU

AD-287616.1
usgsguauCfaAfAfAfccucau
108
asGfsacau(Ggn)agguuuUfgAfua
408
ACUGGUAUCAAAACCUCA
708

gucuL96

ccasgsu

UGUCU

AD-287627.1
csuscugcCfcAfGfUfuuuugu
109
asUfsucac(Agn)aaaacuGfgGfca
409
GUCUCUGCCCAGUUUUUG
709

gaauL96

gagsasc

UGAAU

AD-287632.1
ususuuugUfgAfAfUfacuggg
110
usGfsaacc(Cgn)aguauuCfaCfaa
410
AGUUUUUGUGAAUACUGG
710

uucaL96

aaascsu

GUUCA

AD-287633.1
ususgugaAfuAfCfUfggguuc
111
usGfsguga(Agn)cccaguAfuUfca
411
UUUUGUGAAUACUGGGUU
711

accaL96

caasasa

CACCA

AD-287634.1
usgsugaaUfaCfUfGfgguuca
112
usUfsggug(Agn)acccagUfaUfuc
412
UUUGUGAAUACUGGGUUC
712

ccaaL96

acasasa

ACCAA

AD-287636.1
usgsaauaCfuGfGfGfuucacc
113
usUfsuugg(Tgn)gaacccAfgUfau
413
UGUGAAUACUGGGUUCAC
713

aaaaL96

ucascsa

CAAAA

AD-287637.1
gsasauacUfgGfGfUfucacca
114
usUfsuuug(Ggn)ugaaccCfaGfua
414
GUGAAUACUGGGUUCACC
714

aaaaL96

uucsasc

AAAAA

AD-287638.1
asasuacuGfgGfUfUfcaccaa
115
asUfsuuuu(Ggn)gugaacCfcAfgu
415
UGAAUACUGGGUUCACCA
715

aaauL96

auuscsa

AAAAU

AD-287647.1
asasaaauCfcAfAfGfcacaag
116
usAfsaucu(Tgn)gugcuuGfgAfuu
416
CCAAAAAUCCAAGCACAA
716

auuaL96

uuusgsg

GAUUA

AD-287648.1
asasaaucCfaAfGfCfacaaga
117
asUfsaauc(Tgn)ugugcuUfgGfau
417
CAAAAAUCCAAGCACAAG
717

uuauL96

uuususg

AUUAU

AD-287651.1
asgscacaAfgAfUfUfauggcc
118
usAfscagg(Cgn)cauaauCfuUfgu
418
CAAGCACAAGAUUAUGGC
718

uguaL96

gcususg

CUGUA

AD-287652.1
gscsacaaGfaUfUfAfuggccu
119
asUfsacag(Ggn)ccauaaUfcUfug
419
AAGCACAAGAUUAUGGCC
719

guauL96

ugcsusu

UGUAU

AD-287653.1
csascaagAfuUfAfUfggccug
120
asAfsuaca(Ggn)gccauaAfuCfuu
420
AGCACAAGAUUAUGGCCU
720

uauuL96

gugscsu

GUAUU

AD-287654.1
asasgauuAfuGfGfCfcuguau
121
usCfscaau(Agn)caggccAfuAfau
421
ACAAGAUUAUGGCCUGUA
721

uggaL96

cuusgsu

UUGGA

AD-287655.1
gsasuuauGfgCfCfUfguauug
122
usCfsucca(Agn)uacaggCfcAfua
422
AAGAUUAUGGCCUGUAUU
722

gagaL96

aucsusu

GGAGA

AD-287656.1
ususauggCfcUfGfUfauugga
123
usGfsucuc(Cgn)aauacaGfgCfca
423
GAUUAUGGCCUGUAUUGG
723

gacaL96

uaasusc

AGACA

AD-287658.1
usgsgccuGfuAfUfUfggagac
124
asUfscugu(Cgn)uccaauAfcAfgg
424
UAUGGCCUGUAUUGGAGA
724

agauL96

ccasusa

CAGAU

AD-287659.1
gscscuguAfuUfGfGfagacag
125
usCfsaucu(Ggn)ucuccaAfuAfca
425
UGGCCUGUAUUGGAGACA
725

augaL96

ggcscsa

GAUGA

AD-287679.1
csusgguaUfcAfAfAfaccuca
126
asAfscaug(Agn)gguuuuGfaUfac
426
AACUGGUAUCAAAACCUC
726

uguuL96

cagsusu

AUGUC

AD-287680.1
gsgsuaucAfaAfAfCfcucaug
127
asAfsgaca(Tgn)gagguuUfuGfau
427
CUGGUAUCAAAACCUCAU
727

ucuuL96

accsasg

GUCUC

AD-287683.1
uscsaaaaCfcUfCfAfugucuc
128
asGfscaga(Ggn)acaugaGfgUfuu
428
UAUCAAAACCUCAUGUCU
728

ugcuL96

ugasusa

CUGCC

AD-287690.1
cscscaguUfuUfUfGfugaaua
129
asCfsagua(Tgn)ucacaaAfaAfcu
429
UGCCCAGUUUUUGUGAAU
729

cuguL96

gggscsa

ACUGG

AD-287693.1
ususuuguGfaAfUfAfcugggu
130
asUfsgaac(Cgn)caguauUfcAfca
430
GUUUUUGUGAAUACUGGG
730

ucauL96

aaasasc

UUCAC

AD-287694.1
ususugugAfaUfAfCfuggguu
131
asGfsugaa(Cgn)ccaguaUfuCfac
431
UUUUUGUGAAUACUGGGU
731

cacuL96

aaasasa

UCACC

AD-287695.1
asusacugGfgUfUfCfaccaaa
132
asAfsuuuu(Tgn)ggugaaCfcCfag
432
GAAUACUGGGUUCACCAA
732

aauuL96

uaususc

AAAUC

AD-287696.1
usascuggGfuUfCfAfccaaaa
133
asGfsauuu(Tgn)uggugaAfcCfca
433
AAUACUGGGUUCACCAAA
733

aucuL96

guasusu

AAUCC

AD-287700.1
csasccaaAfaAfUfCfcaagca
134
asUfsugug(Cgn)uuggauUfuUfug
434
UUCACCAAAAAUCCAAGC
734

caauL96

gugsasa

ACAAG

AD-287701.1
asasauccAfaGfCfAfcaagau
135
asAfsuaau(Cgn)uugugcUfuGfga
435
AAAAAUCCAAGCACAAGA
735

uauuL96

uuususu

UUAUG

AD-287702.1
asasuccaAfgCfAfCfaagauu
136
asCfsauaa(Tgn)cuugugCfuUfgg
436
AAAAUCCAAGCACAAGAU
736

auguL96

auususu

UAUGG

AD-287703.1
asusccaaGfcAfCfAfagauua
137
asCfscaua(Agn)ucuuguGfcUfug
437
AAAUCCAAGCACAAGAUU
737

ugguL96

gaususu

AUGGC

AD-287704.1
uscscaagCfaCfAfAfgauuau
138
asGfsccau(Agn)aucuugUfgCfuu
438
AAUCCAAGCACAAGAUUA
738

ggcuL96

ggasusu

UGGCC

AD-287706.1
ascsaagaUfuAfUfGfgccugu
139
asAfsauac(Agn)ggccauAfaUfcu
439
GCACAAGAUUAUGGCCUG
739

auuuL96

ugusgsc

UAUUG

AD-287707.1
csasagauUfaUfGfGfccugua
140
asCfsaaua(Cgn)aggccaUfaAfuc
440
CACAAGAUUAUGGCCUGU
740

uuguL96

uugsusg

AUUGG

AD-287709.1
asusuaugGfcCfUfGfuauugg
141
asUfscucc(Agn)auacagGfcCfau
441
AGAUUAUGGCCUGUAUUG
741

agauL96

aauscsu

GAGAC

AD-287711.1
gsgsccugUfaUfUfGfgagaca
142
asAfsucug(Tgn)cuccaaUfaCfag
442
AUGGCCUGUAUUGGAGAC
742

gauuL96

gccsasu

AGAUG

AD-287724.1
gsusaagaAfgUfCfUfgauaga
143
usCfscauc(Tgn)aucagaCfuUfcu
443
UCGUAAGAAGUCUGAUAG
743

uggaL96

uacsgsa

AUGGA

AD-287725.1
usasagaaGfuCfUfGfauagau
144
usUfsccau(Cgn)uaucagAfcUfuc
444
CGUAAGAAGUCUGAUAGA
744

ggaaL96

uuascsg

UGGAA

AD-287726.1
asasgaagUfcUfGfAfuagaug
145
asUfsucca(Tgn)cuaucaGfaCfuu
445
GUAAGAAGUCUGAUAGAU
745

gaauL96

cuusasc

GGAAU

AD-287727.1
asgsaaguCfuGfAfUfagaugg
146
usAfsuucc(Agn)ucuaucAfgAfcu
446
UAAGAAGUCUGAUAGAUG
746

aauaL96

ucususa

GAAUA

AD-287728.1
asasgucuGfaUfAfGfauggaa
147
asGfsuauu(Cgn)caucuaUfcAfga
447
AGAAGUCUGAUAGAUGGA
747

uacuL96

cuuscsu

AUACU

AD-287735.1
asgsauggAfaUfAfCfuuacca
148
usUfsauug(Ggn)uaaguaUfuCfca
448
AUAGAUGGAAUACUUACC
748

auaaL96

ucusasu

AAUAA

AD-287736.1
asusggaaUfaCfUfUfaccaau
149
usCfsuuau(Tgn)gguaagUfaUfuc
449
AGAUGGAAUACUUACCAA
749

aagaL96

causcsu

UAAGA

AD-287737.1
usgsgaauAfcUfUfAfccaaua
150
usUfscuua(Tgn)ugguaaGfuAfuu
450
GAUGGAAUACUUACCAAU
750

agaaL96

ccasusc

AAGAA

AD-287738.1
gsgsaauaCfuUfAfCfcaauaa
151
usUfsucuu(Agn)uugguaAfgUfau
451
AUGGAAUACUUACCAAUA
751

gaaaL96

uccsasu

AGAAA

AD-287739.1
gsasauacUfuAfCfCfaauaag
152
usUfsuucu(Tgn)auugguAfaGfua
452
UGGAAUACUUACCAAUAA
752

aaaaL96

uucscsa

GAAAA

AD-287740.1
asasuacuUfaCfCfAfauaaga
153
asUfsuuuc(Tgn)uauuggUfaAfgu
453
GGAAUACUUACCAAUAAG
753

aaauL96

auuscsc

AAAAU

AD-287778.1
ususucuuCfcUfGfAfacgcgc
154
usGfsaggc(Ggn)cguucaGfgAfag
454
AGUUUCUUCCUGAACGCG
754

cucaL96

aaascsu

CCUCA

AD-287793.1
gsasuuuuAfaAfUfCfguaugc
155
usUfscugc(Agn)uacgauUfuAfaa
455
GCGAUUUUAAAUCGUAUG
755

agaaL96

aucsgsc

CAGAA

AD-287795.1
ususuuaaAfuCfGfUfaugcag
156
usAfsuucu(Ggn)cauacgAfuUfua
456
GAUUUUAAAUCGUAUGCA
756

aauaL96

aaasusc

GAAUA

AD-287798.1
asasaucgUfaUfGfCfagaaua
157
usGfsaaua(Tgn)ucugcaUfaCfga
457
UUAAAUCGUAUGCAGAAU
757

uucaL96

uuusasa

AUUCA

AD-287800.1
asuscguaUfgCfAfGfaauauu
158
asUfsugaa(Tgn)auucugCfaUfac
458
AAAUCGUAUGCAGAAUAU
758

caauL96

gaususu

UCAAU

AD-287801.1
uscsguauGfcAfGfAfauauuc
159
asAfsuuga(Agn)uauucuGfcAfua
459
AAUCGUAUGCAGAAUAUU
759

aauuL96

cgasusu

CAAUU

AD-287802.1
csgsuaugCfaGfAfAfuauuca
160
asAfsauug(Agn)auauucUfgCfau
460
AUCGUAUGCAGAAUAUUC
760

auuuL96

acgsasu

AAUUU

AD-287804.1
asusgcagAfaUfAfUfucaauu
161
usUfscaaa(Tgn)ugaauaUfuCfug
461
GUAUGCAGAAUAUUCAAU
761

ugaaL96

causasc

UUGAA

AD-287810.1
gsasagucUfgAfUfAfgaugga
162
asUfsauuc(Cgn)aucuauCfaGfac
462
AAGAAGUCUGAUAGAUGG
762

auauL96

uucsusu

AAUAC

AD-287811.1
uscsugauAfgAfUfGfgaauac
163
asUfsaagu(Agn)uuccauCfuAfuc
463
AGUCUGAUAGAUGGAAUA
763

uuauL96

agascsu

CUUAC

AD-287813.1
gsasuggaAfuAfCfUfuaccaa
164
asUfsuauu(Ggn)guaaguAfuUfcc
464
UAGAUGGAAUACUUACCA
764

uaauL96

aucsusa

AUAAG

AD-287837.1
ascsagaaGfuUfUfCfuuccug
165
asGfsuuca(Ggn)gaagaaAfcUfuc
465
CUACAGAAGUUUCUUCCU
765

aacuL96

ugusasg

GAACG

AD-287839.1
asgsaaguUfuCfUfUfccugaa
166
asGfscguu(Cgn)aggaagAfaAfcu
466
ACAGAAGUUUCUUCCUGA
766

cgcuL96

ucusgsu

ACGCG

AD-287841.1
asasguuuCfuUfCfCfugaacg
167
asGfscgcg(Tgn)ucaggaAfgAfaa
467
AGAAGUUUCUUCCUGAAC
767

cgcuL96

cuuscsu

GCGCC

AD-287842.1
gsusuucuUfcCfUfGfaacgcg
168
asAfsggcg(Cgn)guucagGfaAfga
468
AAGUUUCUUCCUGAACGC
768

ccuuL96

aacsusu

GCCUC

AD-287844.1
uscsuuccUfgAfAfCfgcgccu
169
asCfsugag(Ggn)cgcguuCfaGfga
469
UUUCUUCCUGAACGCGCC
769

caguL96

agasasa

UCAGC

AD-287854.1
gscsagaaUfaUfUfCfaauuug
170
asCfsuuca(Agn)auugaaUfaUfuc
470
AUGCAGAAUAUUCAAUUU
770

aaguL96

ugcsasu

GAAGC

AD-287856.1
asasuauuCfaAfUfUfugaagc
171
asAfscugc(Tgn)ucaaauUfgAfau
471
AGAAUAUUCAAUUUGAAG
771

aguuL96

auuscsu

CAGUG

AD-287857.1
usasuucaAfuUfUfGfaagcag
172
asCfscacu(Ggn)cuucaaAfuUfga
472
AAUAUUCAAUUUGAAGCA
772

ugguL96

auasusu

GUGGU

AD-287893.1
gsasuguaUfgCfAfUfgauaau
173
usAfsucau(Tgn)aucaugCfaUfac
473
GAGAUGUAUGCAUGAUAA
773

gauaL96

aucsusc

UGAUA

AD-287917.1
csasaugcUfgCfAfAfagcuuu
174
asAfsauaa(Agn)gcuuugCfaGfca
474
AUCAAUGCUGCAAAGCUU
774

auuuL96

uugsasu

UAUUU

AD-287924.1
asasagcuUfuAfUfUfucacau
175
asAfsaaau(Ggn)ugaaauAfaAfgc
475
GCAAAGCUUUAUUUCACA
775

uuuuL96

uuusgsc

UUUUU

AD-287934.1
asasuuugAfaGfCfAfgugguu
176
asGfsccaa(Cgn)cacugcUfuCfaa
476
UCAAUUUGAAGCAGUGGU
776

ggcuL96

auusgsa

UGGCC

AD-287969.1
asasugcuGfcAfAfAfgcuuua
177
gsAfsaaua(Agn)agcuuuGfcAfgc
477
UCAAUGCUGCAAAGCUUU
777

uuucL96

auusgsa

AUUUC

AD-287970.1
usgscugcAfaAfGfCfuuuauu
178
asUfsgaaa(Tgn)aaagcuUfuGfca
478
AAUGCUGCAAAGCUUUAU
778

ucauL96

gcasusu

UUCAC

AD-287982.1
asasaaacAfuUfGfGfuuuggc
179
usAfsgugc(Cgn)aaaccaAfuGfuu
479
UUAAAAACAUUGGUUUGG
779

acuaL96

uuusasa

CACUA

AD-288005.1
asgsauuaAfuUfAfCfcugucu
180
asGfsgaag(Agn)cagguaAfuUfaa
480
CAAGAUUAAUUACCUGUC
780

uccuL96

ucususg

UUCCU

AD-288006.1
asusuaauUfaCfCfUfgucuuc
181
asCfsagga(Agn)gacaggUfaAfuu
481
AGAUUAAUUACCUGUCUU
781

cuguL96

aauscsu

CCUGU

AD-288008.1
usasauuaCfcUfGfUfcuuccu
182
asAfsacag(Ggn)aagacaGfgUfaa
482
AUUAAUUACCUGUCUUCC
782

guuuL96

uuasasu

UGUUU

AD-288010.1
usasccugUfcUfUfCfcuguuu
183
usGfsagaa(Agn)caggaaGfaCfag
483
AUUACCUGUCUUCCUGUU
783

cucaL96

guasasu

UCUCA

AD-288011.1
ascscuguCfuUfCfCfuguuuc
184
usUfsgaga(Agn)acaggaAfgAfca
484
UUACCUGUCUUCCUGUUU
784

ucaaL96

ggusasa

CUCAA

AD-288015.1
uscsuuccUfgUfUfUfcucaag
185
usAfsuucu(Tgn)gagaaaCfaGfga
485
UGUCUUCCUGUUUCUCAA
785

aauaL96

agascsa

GAAUA

AD-288046.1
ususuuccUfuUfCfAfugccuc
186
usUfsaaga(Ggn)gcaugaAfaGfga
486
GUUUUUCCUUUCAUGCCU
786

uuaaL96

aaasasc

CUUAA

AD-288049.1
uscscuuuCfaUfGfCfcucuua
187
usUfsuuua(Agn)gaggcaUfgAfaa
487
UUUCCUUUCAUGCCUCUU
787

aaaaL96

ggasasa

AAAAA

AD-288050.1
csusuucaUfgCfCfUfcuuaaa
188
asGfsuuuu(Tgn)aagaggCfaUfga
488
UCCUUUCAUGCCUCUUAA
788

aacuL96

aagsgsa

AAACU

AD-288076.1
asasaacaUfuGfGfUfuuggca
189
asUfsagug(Cgn)caaaccAfaUfgu
489
UAAAAACAUUGGUUUGGC
789

cuauL96

uuususa

ACUAG

AD-288092.1
gsasuuaaUfuAfCfCfugucuu
190
asAfsggaa(Ggn)acagguAfaUfua
490
AAGAUUAAUUACCUGUCU
790

ccuuL96

aucsusu

UCCUG

AD-288094.1
ususaccuGfuCfUfUfccuguu
191
asAfsgaaa(Cgn)aggaagAfcAfgg
491
AAUUACCUGUCUUCCUGU
791

ucuuL96

uaasusu

UUCUC

AD-288095.1
cscsugucUfuCfCfUfguuucu
192
asUfsugag(Agn)aacaggAfaGfac
492
UACCUGUCUUCCUGUUUC
792

caauL96

aggsusa

UCAAG

AD-288108.1
gsgsucugUfuUfUfUfccuuuc
193
asCfsauga(Agn)aggaaaAfaCfag
493
UAGGUCUGUUUUUCCUUU
793

auguL96

accsusa

CAUGC

AD-288109.1
gsuscuguUfuUfUfCfcuuuca
194
asGfscaug(Agn)aaggaaAfaAfca
494
AGGUCUGUUUUUCCUUUC
794

ugcuL96

gacscsu

AUGCC

AD-288173.1
ususauuuAfcAfCfAfgggaag
195
asAfsaccu(Tgn)cccuguGfuAfaa
495
ACUUAUUUACACAGGGAA
795

guuuL96

uaasgsu

GGUUU

AD-288174.1
usasuuuaCfaCfAfGfggaagg
196
usAfsaacc(Tgn)ucccugUfgUfaa
496
CUUAUUUACACAGGGAAG
796

uuuaL96

auasasg

GUUUA

AD-288176.1
ususacacAfgGfGfAfagguuu
197
usCfsuuaa(Agn)ccuuccCfuGfug
497
AUUUACACAGGGAAGGUU
797

aagaL96

uaasasu

UAAGA

AD-288177.1
ascsacagGfgAfAfGfguuuaa
198
asGfsucuu(Agn)aaccuuCfcCfug
498
UUACACAGGGAAGGUUUA
798

gacuL96

ugusasa

AGACU

AD-288180.1
gsgsgaagGfuUfUfAfagacug
199
usGfsaaca(Ggn)ucuuaaAfcCfuu
499
CAGGGAAGGUUUAAGACU
799

uucaL96

cccsusg

GUUCA

AD-288181.1
gsgsaaggUfuUfAfAfgacugu
200
usUfsgaac(Agn)gucuuaAfaCfcu
500
AGGGAAGGUUUAAGACUG
800

ucaaL96

uccscsu

UUCAA

AD-288183.1
asgsguuuAfaGfAfCfuguuca
201
usAfscuug(Agn)acagucUfuAfaa
501
GAAGGUUUAAGACUGUUC
801

aguaL96

ccususc

AAGUA

AD-288184.1
ususuaagAfcUfGfUfucaagu
202
usGfscuac(Tgn)ugaacaGfuCfuu
502
GGUUUAAGACUGUUCAAG
802

agcaL96

aaascsc

UAGCA

AD-288186.1
usasagacUfgUfUfCfaaguag
203
asAfsugcu(Agn)cuugaaCfaGfuc
503
UUUAAGACUGUUCAAGUA
803

cauuL96

uuasasa

GCAUU

AD-288187.1
gsascuguUfcAfAfGfuagcau
204
usGfsgaau(Ggn)cuacuuGfaAfca
504
AAGACUGUUCAAGUAGCA
804

uccaL96

gucsusu

UUCCA

AD-288188.1
ascsuguuCfaAfGfUfagcauu
205
usUfsggaa(Tgn)gcuacuUfgAfac
505
AGACUGUUCAAGUAGCAU
805

ccaaL96

aguscsu

UCCAA

AD-288189.1
csusguucAfaGfUfAfgcauuc
206
asUfsugga(Agn)ugcuacUfuGfaa
506
GACUGUUCAAGUAGCAUU
806

caauL96

cagsusc

CCAAU

AD-288190.1
gsusucaaGfuAfGfCfauucca
207
asGfsauug(Ggn)aaugcuAfcUfug
507
CUGUUCAAGUAGCAUUCC
807

aucuL96

aacsasg

AAUCU

AD-288192.1
csasaguaGfcAfUfUfccaauc
208
usAfscaga(Tgn)uggaauGfcUfac
508
UUCAAGUAGCAUUCCAAU
808

uguaL96

uugsasa

CUGUA

AD-288193.1
usasgcauUfcCfAfAfucugua
209
usGfsgcua(Cgn)agauugGfaAfug
509
AGUAGCAUUCCAAUCUGU
809

gccaL96

cuascsu

AGCCA

AD-288194.1
asgscauuCfcAfAfUfcuguag
210
asUfsggcu(Agn)cagauuGfgAfau
510
GUAGCAUUCCAAUCUGUA
810

ccauL96

gcusasc

GCCAU

AD-288227.1
asascuuaUfuUfAfCfacaggg
211
asCfsuucc(Cgn)uguguaAfaUfaa
511
AGAACUUAUUUACACAGG
811

aaguL96

guuscsu

GAAGG

AD-288229.1
usascacaGfgGfAfAfgguuua
212
asUfscuua(Agn)accuucCfcUfgu
512
UUUACACAGGGAAGGUUU
812

agauL96

guasasa

AAGAC

AD-288230.1
csascaggGfaAfGfGfuuuaag
213
asAfsgucu(Tgn)aaaccuUfcCfcu
513
UACACAGGGAAGGUUUAA
813

acuuL96

gugsusa

GACUG

AD-288231.1
asgsggaaGfgUfUfUfaagacu
214
asAfsacag(Tgn)cuuaaaCfcUfuc
514
ACAGGGAAGGUUUAAGAC
814

guuuL96

ccusgsu

UGUUC

AD-288232.1
gsasagguUfuAfAfGfacuguu
215
asUfsugaa(Cgn)agucuuAfaAfcc
515
GGGAAGGUUUAAGACUGU
815

caauL96

uucscsc

UCAAG

AD-288233.1
gsgsuuuaAfgAfCfUfguucaa
216
asUfsacuu(Ggn)aacaguCfuUfaa
516
AAGGUUUAAGACUGUUCA
816

guauL96

accsusu

AGUAG

AD-288235.1
asasgacuGfuUfCfAfaguagc
217
asAfsaugc(Tgn)acuugaAfcAfgu
517
UUAAGACUGUUCAAGUAG
817

auuuL96

cuusasa

CAUUC

AD-288236.1
asgsacugUfuCfAfAfguagca
218
asGfsaaug(Cgn)uacuugAfaCfag
518
UAAGACUGUUCAAGUAGC
818

uucuL96

ucususa

AUUCC

AD-288237.1
usgsuucaAfgUfAfGfcauucc
219
asAfsuugg(Agn)augcuaCfuUfga
519
ACUGUUCAAGUAGCAUUC
819

aauuL96

acasgsu

CAAUC

AD-288238.1
ususcaagUfaGfCfAfuuccaa
220
asAfsgauu(Ggn)gaaugcUfaCfuu
520
UGUUCAAGUAGCAUUCCA
820

ucuuL96

gaascsa

AUCUG

AD-288290.1
gscsagcuUfuAfUfCfucaacc
221
usCfscagg(Tgn)ugagauAfaAfgc
521
AGGCAGCUUUAUCUCAAC
821

uggaL96

ugcscsu

CUGGA

AD-288316.1
asgscauuUfgAfAfAfgauuuc
222
usAfsggga(Agn)aucuuuCfaAfau
522
UCAGCAUUUGAAAGAUUU
822

ccuaL96

gcusgsa

CCCUA

AD-288318.1
usgsaaagAfuUfUfCfccuagc
223
asGfsaggc(Tgn)agggaaAfuCfuu
523
UUUGAAAGAUUUCCCUAG
823

cucuL96

ucasasa

CCUCU

AD-288319.1
gsasaagaUfuUfCfCfcuagcc
224
asAfsgagg(Cgn)uagggaAfaUfcu
524
UUGAAAGAUUUCCCUAGC
824

ucuuL96

uucsasa

CUCUU

AD-288324.1
ususcccuAfgCfCfUfcuuccu
225
asAfsaaag(Ggn)aagaggCfuAfgg
525
AUUUCCCUAGCCUCUUCC
825

uuuuL96

gaasasu

UUUUU

AD-288337.1
asasgaacAfcAfGfAfaugagu
226
asUfsgcac(Tgn)cauucuGfuGfuu
526
ACAAGAACACAGAAUGAG
826

gcauL96

cuusgsu

UGCAC

AD-288338.1
gsasacacAfgAfAfUfgagugc
227
asUfsgugc(Agn)cucauuCfuGfug
527
AAGAACACAGAAUGAGUG
827

acauL96

uucsusu

CACAG

AD-288339.1
asascacaGfaAfUfGfagugca
228
asCfsugug(Cgn)acucauUfcUfgu
528
AGAACACAGAAUGAGUGC
828

caguL96

guuscsu

ACAGC

AD-288340.1
csasgaauGfaGfUfGfcacagc
229
asUfsuagc(Tgn)gugcacUfcAfuu
529
CACAGAAUGAGUGCACAG
829

uaauL96

cugsusg

CUAAG

AD-288341.1
gsasaugaGfuGfCfAfcagcua
230
asUfscuua(Ggn)cugugcAfcUfca
530
CAGAAUGAGUGCACAGCU
830

agauL96

uucsusg

AAGAG

AD-288366.1
ususcagcAfuUfUfGfaaagau
231
asGfsaaau(Cgn)uuucaaAfuGfcu
531
GAUUCAGCAUUUGAAAGA
831

uucuL96

gaasusc

UUUCC

AD-288369.1
csasuuugAfaAfGfAfuuuccc
232
asCfsuagg(Ggn)aaaucuUfuCfaa
532
AGCAUUUGAAAGAUUUCC
832

uaguL96

augscsu

CUAGC

AD-288370.1
asusuugaAfaGfAfUfuucccu
233
asGfscuag(Ggn)gaaaucUfuUfca
533
GCAUUUGAAAGAUUUCCC
833

agcuL96

aausgsc

UAGCC

AD-288391.1
asasaacgGfuGfCfAfacucua
234
asGfsaaua(Ggn)aguugcAfcCfgu
534
CCAAAACGGUGCAACUCU
834

uucuL96

uuusgsg

AUUCU

AD-288393.1
gsgsugcaAfcUfCfUfauucug
235
asGfsucca(Ggn)aauagaGfuUfgc
535
ACGGUGCAACUCUAUUCU
835

gacuL96

accsgsu

GGACU

AD-288394.1
gsusgcaaCfuCfUfAfuucugg
236
asAfsgucc(Agn)gaauagAfgUfug
536
CGGUGCAACUCUAUUCUG
836

acuuL96

cacscsg

GACUU

AD-288395.1
usgscaacUfcUfAfUfucugga
237
asAfsaguc(Cgn)agaauaGfaGfuu
537
GGUGCAACUCUAUUCUGG
837

cuuuL96

gcascsc

ACUUU

AD-288396.1
gscsaacuCfuAfUfUfcuggac
238
usAfsaagu(Cgn)cagaauAfgAfgu
538
GUGCAACUCUAUUCUGGA
838

uuuaL96

ugcsasc

CUUUA

AD-288397.1
csasacucUfaUfUfCfuggacu
239
asUfsaaag(Tgn)ccagaaUfaGfag
539
UGCAACUCUAUUCUGGAC
839

uuauL96

uugscsa

UUUAU

AD-288398.1
asascucuAfuUfCfUfggacuu
240
asAfsuaaa(Ggn)uccagaAfuAfga
540
GCAACUCUAUUCUGGACU
840

uauuL96

guusgsc

UUAUU

AD-288402.1
asusucugGfaCfUfUfuauuac
241
usCfsaagu(Agn)auaaagUfcCfag
541
CUAUUCUGGACUUUAUUA
841

uugaL96

aausasg

CUUGA

AD-288427.1
asgsuccaCfcAfAfAfagugga
242
asGfsgguc(Cgn)acuuuuGfgUfgg
542
GAAGUCCACCAAAAGUGG
842

cccuL96

acususc

ACCCU

AD-288430.1
csasccaaAfaGfUfGfgacccu
243
asUfsagag(Ggn)guccacUfuUfug
543
UCCACCAAAAGUGGACCC
843

cuauL96

gugsgsa

UCUAU

AD-288431.1
ascscaaaAfgUfGfGfacccuc
244
usAfsuaga(Ggn)gguccaCfuUfuu
544
CCACCAAAAGUGGACCCU
844

uauaL96

ggusgsg

CUAUA

AD-288432.1
cscsaaaaGfuGfGfAfcccucu
245
asUfsauag(Agn)ggguccAfcUfuu
545
CACCAAAAGUGGACCCUC
845

auauL96

uggsusg

UAUAU

AD-288433.1
csasaaagUfgGfAfCfccucua
246
asAfsuaua(Ggn)agggucCfaCfuu
546
ACCAAAAGUGGACCCUCU
846

uauuL96

uugsgsu

AUAUU

AD-288434.1
asasaaguGfgAfCfCfcucuau
247
asAfsauau(Agn)gaggguCfcAfcu
547
CCAAAAGUGGACCCUCUA
847

auuuL96

uuusgsg

UAUUU

AD-288466.1
asasacggUfgCfAfAfcucuau
248
asAfsgaau(Agn)gaguugCfaCfcg
548
CAAAACGGUGCAACUCUA
848

ucuuL96

uuususg

UUCUG

AD-288467.1
asascgguGfcAfAfCfucuauu
249
asCfsagaa(Tgn)agaguuGfcAfcc
549
AAAACGGUGCAACUCUAU
849

cuguL96

guususu

UCUGG

AD-288470.1
usasuucuGfgAfCfUfuuauua
250
asAfsagua(Agn)uaaaguCfcAfga
550
UCUAUUCUGGACUUUAUU
850

cuuuL96

auasgsa

ACUUG

AD-288491.1
asasagugGfaCfCfCfucuaua
251
gsAfsaaua(Tgn)agagggUfcCfac
551
CAAAAGUGGACCCUCUAU
851

uuucL96

uuususg

AUUUC

AD-288492.1
asasguggAfcCfCfUfcuauau
252
asGfsaaau(Agn)uagaggGfuCfca
552
AAAAGUGGACCCUCUAUA
852

uucuL96

cuususu

UUUCC

AD-288561.1
asusguuuAfgAfCfAfauuuua
253
asGfsccua(Agn)aauuguCfuAfaa
553
AGAUGUUUAGACAAUUUU
853

ggcuL96

causcsu

AGGCU

AD-288562.1
gsusuuagAfcAfAfUfuuuagg
254
usGfsagcc(Tgn)aaaauuGfuCfua
554
AUGUUUAGACAAUUUUAG
854

cucaL96

aacsasu

GCUCA

AD-288563.1
ususuagaCfaAfUfUfuuaggc
255
usUfsgagc(Cgn)uaaaauUfgUfcu
555
UGUUUAGACAAUUUUAGG
855

ucaaL96

aaascsa

CUCAA

AD-288564.1
ususagacAfaUfUfUfuaggcu
256
usUfsugag(Cgn)cuaaaaUfuGfuc
556
GUUUAGACAAUUUUAGGC
856

caaaL96

uaasasc

UCAAA

AD-288565.1
usasgacaAfuUfUfUfaggcuc
257
usUfsuuga(Ggn)ccuaaaAfuUfgu
557
UUUAGACAAUUUUAGGCU
857

aaaaL96

cuasasa

CAAAA

AD-288566.1
asgsacaaUfuUfUfAfggcuca
258
usUfsuuug(Agn)gccuaaAfaUfug
558
UUAGACAAUUUUAGGCUC
858

aaaaL96

ucusasa

AAAAA

AD-288567.1
gsascaauUfuUfAfGfgcucaa
259
asUfsuuuu(Ggn)agccuaAfaAfuu
559
UAGACAAUUUUAGGCUCA
859

aaauL96

gucsusa

AAAAU

AD-288572.1
ususaggcUfcAfAfAfaauuaa
260
asGfscuuu(Agn)auuuuuGfaGfcc
560
UUUUAGGCUCAAAAAUUA
860

agcuL96

uaasasa

AAGCU

AD-288573.1
usasggcuCfaAfAfAfauuaaa
261
usAfsgcuu(Tgn)aauuuuUfgAfgc
561
UUUAGGCUCAAAAAUUAA
861

gcuaL96

cuasasa

AGCUA

AD-288574.1
asgsgcucAfaAfAfAfuuaaag
262
usUfsagcu(Tgn)uaauuuUfuGfag
562
UUAGGCUCAAAAAUUAAA
862

cuaaL96

ccusasa

GCUAA

AD-288575.1
gscsucaaAfaAfUfUfaaagcu
263
usGfsuuag(Cgn)uuuaauUfuUfug
563
AGGCUCAAAAAUUAAAGC
863

aacaL96

agcscsu

UAACA

AD-288576.1
uscsaaaaAfuUfAfAfagcuaa
264
usGfsuguu(Agn)gcuuuaAfuUfuu
564
GCUCAAAAAUUAAAGCUA
864

cacaL96

ugasgsc

ACACA

AD-288613.1
ususcauaUfaUfCfCfuugguc
265
asUfsggga(Cgn)caaggaUfaUfau
565
CUUUCAUAUAUCCUUGGU
865

ccauL96

gaasasg

CCCAG

AD-288620.1
gsasuguuUfaGfAfCfaauuuu
266
asCfscuaa(Agn)auugucUfaAfac
566
GAGAUGUUUAGACAAUUU
866

agguL96

aucsusc

UAGGC

AD-288624.1
gsgscucaAfaAfAfUfuaaagc
267
asUfsuagc(Tgn)uuaauuUfuUfga
567
UAGGCUCAAAAAUUAAAG
867

uaauL96

gccsusa

CUAAC

AD-288625.1
csuscaaaAfaUfUfAfaagcua
268
asUfsguua(Ggn)cuuuaaUfuUfuu
568
GGCUCAAAAAUUAAAGCU
868

acauL96

gagscsc

AACAC

AD-288626.1
csasaaaaUfuAfAfAfgcuaac
269
asUfsgugu(Tgn)agcuuuAfaUfuu
569
CUCAAAAAUUAAAGCUAA
869

acauL96

uugsasg

CACAG

AD-288628.1
asasauuaAfaGfCfUfaacaca
270
usUfsccug(Tgn)guuagcUfuUfaa
570
AAAAAUUAAAGCUAACAC
870

ggaaL96

uuususu

AGGAA

AD-288634.1
usasacacAfgGfAfAfaaggaa
271
asCfsaguu(Cgn)cuuuucCfuGfug
571
GCUAACACAGGAAAAGGA
871

cuguL96

uuasgsc

CUGUA

AD-288637.1
gsgsaaaaGfgAfAfCfuguacu
272
asGfsccag(Tgn)acaguuCfcUfuu
572
CAGGAAAAGGAACUGUAC
872

ggcuL96

uccsusg

UGGCU

AD-288639.1
asasaaggAfaCfUfGfuacugg
273
asUfsagcc(Agn)guacagUfuCfcu
573
GGAAAAGGAACUGUACUG
873

cuauL96

uuuscsc

GCUAU

AD-288642.1
gsgsaacuGfuAfCfUfggcuau
274
usGfsuaau(Agn)gccaguAfcAfgu
574
AAGGAACUGUACUGGCUA
874

uacaL96

uccsusu

UUACA

AD-288696.1
csasggaaAfaGfGfAfacugua
275
asCfsagua(Cgn)aguuccUfuUfuc
575
CACAGGAAAAGGAACUGU
875

cuguL96

cugsusg

ACUGG

AD-288698.1
asgsgaacUfgUfAfCfuggcua
276
asUfsaaua(Ggn)ccaguaCfaGfuu
576
AAAGGAACUGUACUGGCU
876

uuauL96

ccususu

AUUAC

AD-288912.1
csuscccaCfuAfCfAfucaaga
277
usUfsaguc(Tgn)ugauguAfgUfgg
577
GACUCCCACUACAUCAAG
877

cuaaL96

gagsusc

ACUAA

AD-288913.1
uscsccacUfaCfAfUfcaagac
278
asUfsuagu(Cgn)uugaugUfaGfug
578
ACUCCCACUACAUCAAGA
878

uaauL96

ggasgsu

CUAAU

AD-288914.1
cscsacuaCfaUfCfAfagacua
279
asGfsauua(Ggn)ucuugaUfgUfag
579
UCCCACUACAUCAAGACU
879

aucuL96

uggsgsa

AAUCU

AD-288915.1
csascuacAfuCfAfAfgacuaa
280
asAfsgauu(Agn)gucuugAfuGfua
580
CCCACUACAUCAAGACUA
880

ucuuL96

gugsgsg

AUCUU

AD-288916.1
csusacauCfaAfGfAfcuaauc
281
asCfsaaga(Tgn)uagucuUfgAfug
581
CACUACAUCAAGACUAAU
881

uuguL96

uagsusg

CUUGU

AD-288917.1
usascaucAfaGfAfCfuaaucu
282
asAfscaag(Agn)uuagucUfuGfau
582
ACUACAUCAAGACUAAUC
882

uguuL96

guasgsu

UUGUU

AD-288918.1
ascsaucaAfgAfCfUfaaucuu
283
asAfsacaa(Ggn)auuaguCfuUfga
583
CUACAUCAAGACUAAUCU
883

guuuL96

ugusasg

UGUUU

AD-288933.1
gsusguuuUfuCfAfCfauguau
284
usAfsuaau(Agn)caugugAfaAfaa
584
GUGUGUUUUUCACAUGUA
884

uauaL96

cacsasc

UUAUA

AD-288937.1
uscsacauGfuAfUfUfauagaa
285
asGfscauu(Cgn)uauaauAfcAfug
585
UUUCACAUGUAUUAUAGA
885

ugcuL96

ugasasa

AUGCU

AD-288938.1
csascaugUfaUfUfAfuagaau
286
asAfsgcau(Tgn)cuauaaUfaCfau
586
UUCACAUGUAUUAUAGAA
886

gcuuL96

gugsasa

UGCUU

AD-288941.1
gsusauuaUfaGfAfAfugcuuu
287
usGfscaaa(Agn)gcauucUfaUfaa
587
AUGUAUUAUAGAAUGCUU
887

ugcaL96

uacsasu

UUGCA

AD-288943.1
usasuagaAfuGfCfUfuuugca
288
usCfscaug(Cgn)aaaagcAfuUfcu
588
AUUAUAGAAUGCUUUUGC
888

uggaL96

auasasu

AUGGA

AD-288945.1
asgsaaugCfuUfUfUfgcaugg
289
usAfsgucc(Agn)ugcaaaAfgCfau
589
AUAGAAUGCUUUUGCAUG
889

acuaL96

ucusasu

GACUA

AD-288946.1
gsasaugcUfuUfUfGfcaugga
290
asUfsaguc(Cgn)augcaaAfaGfca
590
UAGAAUGCUUUUGCAUGG
890

cuauL96

uucsusa

ACUAU

AD-288947.1
usgscuuuUfgCfAfUfggacua
291
asGfsgaua(Ggn)uccaugCfaAfaa
591
AAUGCUUUUGCAUGGACU
891

uccuL96

gcasusu

AUCCU

AD-288948.1
csusuuugCfaUfGfGfacuauc
292
asGfsagga(Tgn)aguccaUfgCfaa
592
UGCUUUUGCAUGGACUAU
892

cucuL96

aagscsa

CCUCU

AD-288950.1
ususgcauGfgAfCfUfauccuc
293
asCfsaaga(Ggn)gauaguCfcAfug
593
UUUUGCAUGGACUAUCCU
893

uuguL96

caasasa

CUUGU

AD-288951.1
usgscaugGfaCfUfAfuccucu
294
asAfscaag(Agn)ggauagUfcCfau
594
UUUGCAUGGACUAUCCUC
894

uguuL96

gcasasa

UUGUU

AD-288952.1
gscsauggAfcUfAfUfccucuu
295
asAfsacaa(Ggn)aggauaGfuCfca
595
UUGCAUGGACUAUCCUCU
895

guuuL96

ugcsasa

UGUUU

AD-288956.1
gsgsacuaUfcCfUfCfuuguuu
296
asUfsaaaa(Agn)caagagGfaUfag
596
AUGGACUAUCCUCUUGUU
896

uuauL96

uccsasu

UUUAU

AD-288957.1
gsascuauCfcUfCfUfuguuuu
297
asAfsuaaa(Agn)acaagaGfgAfua
597
UGGACUAUCCUCUUGUUU
897

uauuL96

gucscsa

UUAUU

AD-288959.1
csusauccUfcUfUfGfuuuuua
298
usUfsaaua(Agn)aaacaaGfaGfga
598
GACUAUCCUCUUGUUUUU
898

uuaaL96

uagsusc

AUUAA

AD-288981.1
cscscacuAfcAfUfCfaagacu
299
asAfsuuag(Tgn)cuugauGfuAfgu
599
CUCCCACUACAUCAAGAC
899

aauuL96

gggsasg

UAAUC

AD-288982.1
ascsuacaUfcAfAfGfacuaau
300
asAfsagau(Tgn)agucuuGfaUfgu
600
CCACUACAUCAAGACUAA
900

cuuuL96

agusgsg

UCUUG

AD-288996.1
asusgcuuUfuGfCfAfuggacu
301
asGfsauag(Tgn)ccaugcAfaAfag
601
GAAUGCUUUUGCAUGGAC
901

aucuL96

caususc

UAUCC

*Target: HSD17B13

TABLE 8

HSD17B13 ELF7 GNA7 Unmodified Sequence*

SEQ
Position

SEQ
Position

Sense sequence
ID
in
Antisense sequence
ID
in

Duplex Name
5′ to 3′
NO:
NM_178135.4
5′ to 3′
NO:
NM_178135.4

AD-286958.1
AGUACCUCCUCCCUAGGACUA
902
5-25
UAGUCCTAGGGAGGAGGUACUGU
1202
3-25

AD-287001.1
UCCUUCUGCUUCUGAUCACCA
903
85-105
UGGUGATCAGAAGCAGAAGGAUU
1203
83-105

AD-287012.1
CAUCAUCUACUCCUACUUGGA
904
104-124
UCCAAGTAGGAGUAGAUGAUGGU
1204
102-124

AD-287015.1
CUACUCCUACUUGGAGUCGUU
906
110-130
AACGACTCCAAGUAGGAGUAGAU
1206
108-130

AD-287017.1
GUACCUCCUCCCUAGGACUAU
907
6-26
AUAGUCCUAGGGAGGAGGUACUG
1207
4-26

AD-287026.1
ACACAAGGACUGAACCAGAAU
908
25-45
AUUCUGGUUCAGUCCUUGUGUAG
1208
23-45

AD-287059.1
ACCAUCAUCUACUCCUACUUU
909
102-122
AAAGUAGGAGUAGAUGAUGGUGA
1209
100-122

AD-287060.1
CCAUCAUCUACUCCUACUUGU
910
103-123
ACAAGUAGGAGUAGAUGAUGGUG
1210
101-123

AD-287061.1
AUCAUCUACUCCUACUUGGAU
911
105-125
AUCCAAGUAGGAGUAGAUGAUGG
1211
103-125

AD-287062.1
CAUCUACUCCUACUUGGAGUU
912
107-127
AACUCCAAGUAGGAGUAGAUGAU
1212
105-127

AD-287064.1
UACUCCUACUUGGAGUCGUUU
913
111-131
AAACGACUCCAAGUAGGAGUAGA
1213
109-131

AD-287065.1
ACUCCUACUUGGAGUCGUUGU
914
112-132
ACAACGACUCCAAGUAGGAGUAG
1214
110-132

AD-287126.1
UCCUACUUGGAGUCGUUGGUU
916
114-134
AACCAACGACUCCAAGUAGGAGU
1216
112-134

AD-287203.1
GCAGCUGAGUGCCGAAAACUA
920
294-314
UAGUUUTCGGCACUCAGCUGCAG
1220
292-314

AD-287227.1
CUGUGGGAUAUUAAUAAGCGU
923
258-278
ACGCUUAUUAAUAUCCCACAGAA
1223
256-278

AD-287246.1
CUGCAGCUGAGUGCCGAAAAU
924
292-312
AUUUUCGGCACUCAGCUGCAGUU
1224
290-312

AD-287247.1
CAGCUGAGUGCCGAAAACUAU
925
295-315
AUAGUUTUCGGCACUCAGCUGCA
1225
293-315

AD-287248.1
AGCUGAGUGCCGAAAACUAGU
926
296-316
ACUAGUTUUCGGCACUCAGCUGC
1226
294-316

AD-287249.1
GCUGAGUGCCGAAAACUAGGU
927
297-317
ACCUAGTUUUCGGCACUCAGCUG
1227
295-317

AD-287250.1
CUGAGUGCCGAAAACUAGGCU
928
298-318
AGCCUAGUUUUCGGCACUCAGCU
1228
296-318

AD-287251.1
GAGUGCCGAAAACUAGGCGUU
929
300-320
AACGCCTAGUUUUCGGCACUCAG
1229
298-320

AD-287252.1
GUGCCGAAAACUAGGCGUCAU
930
302-322
AUGACGCCUAGUUUUCGGCACUC
1230
300-322

AD-287253.1
GCCGAAAACUAGGCGUCACUU
931
304-324
AAGUGACGCCUAGUUUUCGGCAC
1231
302-324

AD-287254.1
CCGAAAACUAGGCGUCACUGU
932
305-325
ACAGUGACGCCUAGUUUUCGGCA
1232
303-325

AD-287258.1
ACUAGGCGUCACUGCGCAUGU
933
311-331
ACAUGCGCAGUGACGCCUAGUUU
1233
309-331

AD-287288.1
CUAUCGCUCUCUAAAUCAGGU
935
365-385
ACCUGATUUAGAGAGCGAUAGAU
1235
363-385

AD-287290.1
UCGCUCUCUAAAUCAGGUGAA
937
368-388
UUCACCTGAUUUAGAGAGCGAUA
1237
366-388

AD-287293.1
UCUCUAAAUCAGGUGAAGAAA
938
372-392
UUUCUUCACCUGAUUUAGAGAGC
1238
370-392

AD-287316.1
GUGGUGAAUAAUGCUGGGACA
939
414-434
UGUCCCAGCAUUAUUCACCACGA
1239
412-434

AD-287320.1
GAAUAAUGCUGGGACAGUAUA
940
419-439
UAUACUGUCCCAGCAUUAUUCAC
1240
417-439

AD-287321.1
AAUAAUGCUGGGACAGUAUAU
941
420-440
AUAUACTGUCCCAGCAUUAUUCA
1241
418-440

AD-287324.1
GGGACAGUAUAUCCAGCCGAU
942
429-449
AUCGGCTGGAUAUACUGUCCCAG
1242
427-449

AD-287325.1
GACAGUAUAUCCAGCCGAUCU
943
431-451
AGAUCGGCUGGAUAUACUGUCCC
1243
429-451

AD-287326.1
ACAGUAUAUCCAGCCGAUCUU
944
432-452
AAGAUCGGCUGGAUAUACUGUCC
1244
430-452

AD-287327.1
AGUAUAUCCAGCCGAUCUUCU
945
434-454
AGAAGATCGGCUGGAUAUACUGU
1245
432-454

AD-287344.1
AUCUAUCGCUCUCUAAAUCAU
946
363-383
AUGAUUTAGAGAGCGAUAGAUCU
1246
361-383

AD-287346.1
UAUCGCUCUCUAAAUCAGGUU
947
366-386
AACCUGAUUUAGAGAGCGAUAGA
1247
364-386

AD-287347.1
CGCUCUCUAAAUCAGGUGAAU
948
369-389
AUUCACCUGAUUUAGAGAGCGAU
1248
367-389

AD-287366.1
CGUGGUGAAUAAUGCUGGGAU
949
413-433
AUCCCAGCAUUAUUCACCACGAU
1249
411-433

AD-287367.1
UGGUGAAUAAUGCUGGGACAU
950
415-435
AUGUCCCAGCAUUAUUCACCACG
1250
413-435

AD-287370.1
AUGCUGGGACAGUAUAUCCAU
951
424-444
AUGGAUAUACUGUCCCAGCAUUA
1251
422-444

AD-287371.1
UGCUGGGACAGUAUAUCCAGU
952
425-445
ACUGGATAUACUGUCCCAGCAUU
1252
423-445

AD-287372.1
GCUGGGACAGUAUAUCCAGCU
953
426-446
AGCUGGAUAUACUGUCCCAGCAU
1253
424-446

AD-287373.1
CUGGGACAGUAUAUCCAGCCU
954
427-447
AGGCUGGAUAUACUGUCCCAGCA
1254
425-447

AD-287374.1
GGACAGUAUAUCCAGCCGAUU
955
430-450
AAUCGGCUGGAUAUACUGUCCCA
1255
428-450

AD-287375.1
CAGUAUAUCCAGCCGAUCUUU
956
433-453
AAAGAUCGGCUGGAUAUACUGUC
1256
431-453

AD-287391.1
CCAAGGAUGAAGAGAUUACCA
957
460-480
UGGUAATCUCUUCAUCCUUGGUG
1257
458-480

AD-287406.1
UUUGAGGUCAACAUCCUAGGA
959
486-506
UCCUAGGAUGUUGACCUCAAAUG
1259
484-506

AD-287407.1
UGAGGUCAACAUCCUAGGACA
960
488-508
UGUCCUAGGAUGUUGACCUCAAA
1260
486-508

AD-287408.1
GAGGUCAACAUCCUAGGACAU
961
489-509
AUGUCCTAGGAUGUUGACCUCAA
1261
487-509

AD-287411.1
GUCAACAUCCUAGGACAUUUU
963
492-512
AAAAUGTCCUAGGAUGUUGACCU
1263
490-512

AD-287413.1
ACAUCCUAGGACAUUUUUGGA
964
496-516
UCCAAAAAUGUCCUAGGAUGUUG
1264
494-516

AD-287414.1
CAUCCUAGGACAUUUUUGGAU
965
497-517
AUCCAAAAAUGUCCUAGGAUGUU
1265
495-517

AD-287418.1
AGGACAUUUUUGGAUCACAAA
967
503-523
UUUGUGAUCCAAAAAUGUCCUAG
1267
501-523

AD-287419.1
GGACAUUUUUGGAUCACAAAA
968
504-524
UUUUGUGAUCCAAAAAUGUCCUA
1268
502-524

AD-287421.1
UUUUUGGAUCACAAAAGCACU
969
509-529
AGUGCUTUUGUGAUCCAAAAAUG
1269
507-529

AD-287424.1
UGGAUCACAAAAGCACUUCUU
970
513-533
AAGAAGTGCUUUUGUGAUCCAAA
1270
511-533

AD-287427.1
CAAAAGCACUUCUUCCAUCGA
971
520-540
UCGAUGGAAGAAGUGCUUUUGUG
1271
518-540

AD-287428.1
AAAAGCACUUCUUCCAUCGAU
972
521-541
AUCGAUGGAAGAAGUGCUUUUGU
1272
519-541

AD-287453.1
ACCAAGGAUGAAGAGAUUACU
976
459-479
AGUAAUCUCUUCAUCCUUGGUGC
1276
457-479

AD-287462.1
AGACAUUUGAGGUCAACAUCU
977
481-501
AGAUGUTGACCUCAAAUGUCUUG
1277
479-501

AD-287463.1
CAUUUGAGGUCAACAUCCUAU
978
484-504
AUAGGATGUUGACCUCAAAUGUC
1278
482-504

AD-287464.1
AUUUGAGGUCAACAUCCUAGU
979
485-505
ACUAGGAUGUUGACCUCAAAUGU
1279
483-505

AD-287465.1
UUGAGGUCAACAUCCUAGGAU
980
487-507
AUCCUAGGAUGUUGACCUCAAAU
1280
485-507

AD-287466.1
CAACAUCCUAGGACAUUUUUG
981
494-514
CAAAAATGUCCUAGGAUGUUGAC
1281
492-514

AD-287467.1
AACAUCCUAGGACAUUUUUGU
982
495-515
ACAAAAAUGUCCUAGGAUGUUGA
1282
493-515

AD-287468.1
AUCCUAGGACAUUUUUGGAUU
983
498-518
AAUCCAAAAAUGUCCUAGGAUGU
1283
496-518

AD-287469.1
CCUAGGACAUUUUUGGAUCAU
984
500-520
AUGAUCCAAAAAUGUCCUAGGAU
1284
498-520

AD-287470.1
GACAUUUUUGGAUCACAAAAU
985
505-525
AUUUUGTGAUCCAAAAAUGUCCU
1285
503-525

AD-287474.1
GGAUCACAAAAGCACUUCUUU
986
514-534
AAAGAAGUGCUUUUGUGAUCCAA
1286
512-534

AD-287476.1
CACAAAAGCACUUCUUCCAUU
987
518-538
AAUGGAAGAAGUGCUUUUGUGAU
1287
516-538

AD-287477.1
ACAAAAGCACUUCUUCCAUCU
988
519-539
AGAUGGAAGAAGUGCUUUUGUGA
1288
517-539

AD-287478.1
AAAGCACUUCUUCCAUCGAUU
989
522-542
AAUCGATGGAAGAAGUGCUUUUG
1289
520-542

AD-287479.1
GCACUUCUUCCAUCGAUGAUU
990
525-545
AAUCAUCGAUGGAAGAAGUGCUU
1290
523-545

AD-287491.1
AGAAAUCAUGGCCACAUCGUU
991
549-569
AACGAUGUGGCCAUGAUUUCUCU
1291
547-569

AD-287492.1
AAAUCAUGGCCACAUCGUCAU
992
551-571
AUGACGAUGUGGCCAUGAUUUCU
1292
549-571

AD-287517.1
UUCCUUACCUCAUCCCAUAUU
993
601-621
AAUAUGGGAUGAGGUAAGGAAUC
1293
599-621

AD-287520.1
ACCUCAUCCCAUAUUGUUCCA
994
607-627
UGGAACAAUAUGGGAUGAGGUAA
1294
605-627

AD-287521.1
UCAUCCCAUAUUGUUCCAGCA
995
610-630
UGCUGGAACAAUAUGGGAUGAGG
1295
608-630

AD-287524.1
UCCCAUAUUGUUCCAGCAAAU
997
613-633
AUUUGCTGGAACAAUAUGGGAUG
1297
611-633

AD-287525.1
CCCAUAUUGUUCCAGCAAAUU
998
614-634
AAUUUGCUGGAACAAUAUGGGAU
1298
612-634

AD-287537.1
UGUUGGCUUUCACAGAGGUCU
999
641-661
AGACCUCUGUGAAAGCCAACAGC
1299
639-661

AD-287539.1
GGCUUUCACAGAGGUCUGACA
1000
645-665
UGUCAGACCUCUGUGAAAGCCAA
1300
643-665

AD-287543.1
CACAGAGGUCUGACAUCAGAA
1002
651-671
UUCUGATGUCAGACCUCUGUGAA
1302
649-671

AD-287546.1
AGGUCUGACAUCAGAACUUCA
1003
656-676
UGAAGUTCUGAUGUCAGACCUCU
1303
654-676

AD-287568.1
UCCUUACCUCAUCCCAUAUUU
1004
602-622
AAAUAUGGGAUGAGGUAAGGAAU
1304
600-622

AD-287591.1
UUCACAGAGGUCUGACAUCAU
1005
649-669
AUGAUGTCAGACCUCUGUGAAAG
1305
647-669

AD-287610.1
UUGGGAAAAACUGGUAUCAAA
1006
681-701
UUUGAUACCAGUUUUUCCCAAGG
1306
679-701

AD-287616.1
UGGUAUCAAAACCUCAUGUCU
1008
692-712
AGACAUGAGGUUUUGAUACCAGU
1308
690-712

AD-287627.1
CUCUGCCCAGUUUUUGUGAAU
1009
711-731
AUUCACAAAAACUGGGCAGAGAC
1309
709-731

AD-287632.1
UUUUUGUGAAUACUGGGUUCA
1010
721-741
UGAACCCAGUAUUCACAAAAACU
1310
719-741

AD-287636.1
UGAAUACUGGGUUCACCAAAA
1013
727-747
UUUUGGTGAACCCAGUAUUCACA
1313
725-747

AD-287637.1
GAAUACUGGGUUCACCAAAAA
1014
728-748
UUUUUGGUGAACCCAGUAUUCAC
1314
726-748

AD-287647.1
AAAAAUCCAAGCACAAGAUUA
1016
744-764
UAAUCUTGUGCUUGGAUUUUUGG
1316
742-764

AD-287648.1
AAAAUCCAAGCACAAGAUUAU
1017
745-765
AUAAUCTUGUGCUUGGAUUUUUG
1317
743-765

AD-287654.1
AAGAUUAUGGCCUGUAUUGGA
1021
758-778
UCCAAUACAGGCCAUAAUCUUGU
1321
756-778

AD-287655.1
GAUUAUGGCCUGUAUUGGAGA
1022
760-780
UCUCCAAUACAGGCCAUAAUCUU
1322
758-780

AD-287659.1
GCCUGUAUUGGAGACAGAUGA
1025
767-787
UCAUCUGUCUCCAAUACAGGCCA
1325
765-787

AD-287679.1
CUGGUAUCAAAACCUCAUGUU
1026
691-711
AACAUGAGGUUUUGAUACCAGUU
1326
689-711

AD-287680.1
GGUAUCAAAACCUCAUGUCUU
1027
693-713
AAGACATGAGGUUUUGAUACCAG
1327
691-713

AD-287683.1
UCAAAACCUCAUGUCUCUGCU
1028
697-717
AGCAGAGACAUGAGGUUUUGAUA
1328
695-717

AD-287690.1
CCCAGUUUUUGUGAAUACUGU
1029
716-736
ACAGUATUCACAAAAACUGGGCA
1329
714-736

AD-287693.1
UUUUGUGAAUACUGGGUUCAU
1030
722-742
AUGAACCCAGUAUUCACAAAAAC
1330
720-742

AD-287694.1
UUUGUGAAUACUGGGUUCACU
1031
723-743
AGUGAACCCAGUAUUCACAAAAA
1331
721-743

AD-287695.1
AUACUGGGUUCACCAAAAAUU
1032
730-750
AAUUUUTGGUGAACCCAGUAUUC
1332
728-750

AD-287696.1
UACUGGGUUCACCAAAAAUCU
1033
731-751
AGAUUUTUGGUGAACCCAGUAUU
1333
729-751

AD-287700.1
CACCAAAAAUCCAAGCACAAU
1034
740-760
AUUGUGCUUGGAUUUUUGGUGAA
1334
738-760

AD-287701.1
AAAUCCAAGCACAAGAUUAUU
1035
746-766
AAUAAUCUUGUGCUUGGAUUUUU
1335
744-766

AD-287702.1
AAUCCAAGCACAAGAUUAUGU
1036
747-767
ACAUAATCUUGUGCUUGGAUUUU
1336
745-767

AD-287703.1
AUCCAAGCACAAGAUUAUGGU
1037
748-768
ACCAUAAUCUUGUGCUUGGAUUU
1337
746-768

AD-287704.1
UCCAAGCACAAGAUUAUGGCU
1038
749-769
AGCCAUAAUCUUGUGCUUGGAUU
1338
747-769

AD-287706.1
ACAAGAUUAUGGCCUGUAUUU
1039
756-776
AAAUACAGGCCAUAAUCUUGUGC
1339
754-776

AD-287707.1
CAAGAUUAUGGCCUGUAUUGU
1040
757-777
ACAAUACAGGCCAUAAUCUUGUG
1340
755-777

AD-287709.1
AUUAUGGCCUGUAUUGGAGAU
1041
761-781
AUCUCCAAUACAGGCCAUAAUCU
1341
759-781

AD-287711.1
GGCCUGUAUUGGAGACAGAUU
1042
766-786
AAUCUGTCUCCAAUACAGGCCAU
1342
764-786

AD-287724.1
GUAAGAAGUCUGAUAGAUGGA
1043
792-812
UCCAUCTAUCAGACUUCUUACGA
1343
790-812

AD-287726.1
AAGAAGUCUGAUAGAUGGAAU
1045
794-814
AUUCCATCUAUCAGACUUCUUAC
1345
792-814

AD-287727.1
AGAAGUCUGAUAGAUGGAAUA
1046
795-815
UAUUCCAUCUAUCAGACUUCUUA
1346
793-815

AD-287735.1
AGAUGGAAUACUUACCAAUAA
1048
806-826
UUAUUGGUAAGUAUUCCAUCUAU
1348
804-826

AD-287736.1
AUGGAAUACUUACCAAUAAGA
1049
808-828
UCUUAUTGGUAAGUAUUCCAUCU
1349
806-828

AD-287737.1
UGGAAUACUUACCAAUAAGAA
1050
809-829
UUCUUATUGGUAAGUAUUCCAUC
1350
807-829

AD-287738.1
GGAAUACUUACCAAUAAGAAA
1051
810-830
UUUCUUAUUGGUAAGUAUUCCAU
1351
808-830

AD-287739.1
GAAUACUUACCAAUAAGAAAA
1052
811-831
UUUUCUTAUUGGUAAGUAUUCCA
1352
809-831

AD-287740.1
AAUACUUACCAAUAAGAAAAU
1053
812-832
AUUUUCTUAUUGGUAAGUAUUCC
1353
810-832

AD-287778.1
UUUCUUCCUGAACGCGCCUCA
1054
879-899
UGAGGCGCGUUCAGGAAGAAACU
1354
877-899

AD-287798.1
AAAUCGUAUGCAGAAUAUUCA
1057
908-928
UGAAUATUCUGCAUACGAUUUAA
1357
906-928

AD-287800.1
AUCGUAUGCAGAAUAUUCAAU
1058
910-930
AUUGAATAUUCUGCAUACGAUUU
1358
908-930

AD-287801.1
UCGUAUGCAGAAUAUUCAAUU
1059
911-931
AAUUGAAUAUUCUGCAUACGAUU
1359
909-931

AD-287802.1
CGUAUGCAGAAUAUUCAAUUU
1060
912-932
AAAUUGAAUAUUCUGCAUACGAU
1360
910-932

AD-287804.1
AUGCAGAAUAUUCAAUUUGAA
1061
915-935
UUCAAATUGAAUAUUCUGCAUAC
1361
913-935

AD-287810.1
GAAGUCUGAUAGAUGGAAUAU
1062
796-816
AUAUUCCAUCUAUCAGACUUCUU
1362
794-816

AD-287811.1
UCUGAUAGAUGGAAUACUUAU
1063
800-820
AUAAGUAUUCCAUCUAUCAGACU
1363
798-820

AD-287813.1
GAUGGAAUACUUACCAAUAAU
1064
807-827
AUUAUUGGUAAGUAUUCCAUCUA
1364
805-827

AD-287837.1
ACAGAAGUUUCUUCCUGAACU
1065
872-892
AGUUCAGGAAGAAACUUCUGUAG
1365
870-892

AD-287839.1
AGAAGUUUCUUCCUGAACGCU
1066
874-894
AGCGUUCAGGAAGAAACUUCUGU
1366
872-894

AD-287841.1
AAGUUUCUUCCUGAACGCGCU
1067
876-896
AGCGCGTUCAGGAAGAAACUUCU
1367
874-896

AD-287842.1
GUUUCUUCCUGAACGCGCCUU
1068
878-898
AAGGCGCGUUCAGGAAGAAACUU
1368
876-898

AD-287844.1
UCUUCCUGAACGCGCCUCAGU
1069
881-901
ACUGAGGCGCGUUCAGGAAGAAA
1369
879-901

AD-287854.1
GCAGAAUAUUCAAUUUGAAGU
1070
917-937
ACUUCAAAUUGAAUAUUCUGCAU
1370
915-937

AD-287856.1
AAUAUUCAAUUUGAAGCAGUU
1071
921-941
AACUGCTUCAAAUUGAAUAUUCU
1371
919-941

AD-287893.1
GAUGUAUGCAUGAUAAUGAUA
1073
989-1009
UAUCAUTAUCAUGCAUACAUCUC
1373
987-1009

AD-287917.1
CAAUGCUGCAAAGCUUUAUUU
1074
1025-1045
AAAUAAAGCUUUGCAGCAUUGAU
1374
1023-1045

AD-287924.1
AAAGCUUUAUUUCACAUUUUU
1075
1034-1054
AAAAAUGUGAAAUAAAGCUUUGC
1375
1032-1054

AD-287934.1
AAUUUGAAGCAGUGGUUGGCU
1076
928-948
AGCCAACCACUGCUUCAAAUUGA
1376
926-948

AD-287969.1
AAUGCUGCAAAGCUUUAUUUC
1077
1026-1046
GAAAUAAAGCUUUGCAGCAUUGA
1377
1024-1046

AD-287970.1
UGCUGCAAAGCUUUAUUUCAU
1078
1028-1048
AUGAAATAAAGCUUUGCAGCAUU
1378
1026-1048

AD-288005.1
AGAUUAAUUACCUGUCUUCCU
1080
1111-1131
AGGAAGACAGGUAAUUAAUCUUG
1380
1109-1131

AD-288006.1
AUUAAUUACCUGUCUUCCUGU
1081
1113-1133
ACAGGAAGACAGGUAAUUAAUCU
1381
1111-1133

AD-288008.1
UAAUUACCUGUCUUCCUGUUU
1082
1115-1135
AAACAGGAAGACAGGUAAUUAAU
1382
1113-1135

AD-288010.1
UACCUGUCUUCCUGUUUCUCA
1083
1119-1139
UGAGAAACAGGAAGACAGGUAAU
1383
1117-1139

AD-288011.1
ACCUGUCUUCCUGUUUCUCAA
1084
1120-1140
UUGAGAAACAGGAAGACAGGUAA
1384
1118-1140

AD-288015.1
UCUUCCUGUUUCUCAAGAAUA
1085
1125-1145
UAUUCUTGAGAAACAGGAAGACA
1385
1123-1145

AD-288046.1
UUUUCCUUUCAUGCCUCUUAA
1086
1171-1191
UUAAGAGGCAUGAAAGGAAAAAC
1386
1169-1191

AD-288049.1
UCCUUUCAUGCCUCUUAAAAA
1087
1174-1194
UUUUUAAGAGGCAUGAAAGGAAA
1387
1172-1194

AD-288050.1
CUUUCAUGCCUCUUAAAAACU
1088
1176-1196
AGUUUUTAAGAGGCAUGAAAGGA
1388
1174-1196

AD-288076.1
AAAACAUUGGUUUGGCACUAU
1089
1073-1093
AUAGUGCCAAACCAAUGUUUUUA
1389
1071-1093

AD-288092.1
GAUUAAUUACCUGUCUUCCUU
1090
1112-1132
AAGGAAGACAGGUAAUUAAUCUU
1390
1110-1132

AD-288094.1
UUACCUGUCUUCCUGUUUCUU
1091
1118-1138
AAGAAACAGGAAGACAGGUAAUU
1391
1116-1138

AD-288095.1
CCUGUCUUCCUGUUUCUCAAU
1092
1121-1141
AUUGAGAAACAGGAAGACAGGUA
1392
1119-1141

AD-288108.1
GGUCUGUUUUUCCUUUCAUGU
1093
1164-1184
ACAUGAAAGGAAAAACAGACCUA
1393
1162-1184

AD-288109.1
GUCUGUUUUUCCUUUCAUGCU
1094
1165-1185
AGCAUGAAAGGAAAAACAGACCU
1394
1163-1185

AD-288173.1
UUAUUUACACAGGGAAGGUUU
1095
1302-1322
AAACCUTCCCUGUGUAAAUAAGU
1395
1300-1322

AD-288174.1
UAUUUACACAGGGAAGGUUUA
1096
1303-1323
UAAACCTUCCCUGUGUAAAUAAG
1396
1301-1323

AD-288180.1
GGGAAGGUUUAAGACUGUUCA
1099
1313-1333
UGAACAGUCUUAAACCUUCCCUG
1399
1311-1333

AD-288181.1
GGAAGGUUUAAGACUGUUCAA
1100
1314-1334
UUGAACAGUCUUAAACCUUCCCU
1400
1312-1334

AD-288184.1
UUUAAGACUGUUCAAGUAGCA
1102
1320-1340
UGCUACTUGAACAGUCUUAAACC
1402
1318-1340

AD-288187.1
GACUGUUCAAGUAGCAUUCCA
1104
1325-1345
UGGAAUGCUACUUGAACAGUCUU
1404
1323-1345

AD-288188.1
ACUGUUCAAGUAGCAUUCCAA
1105
1326-1346
UUGGAATGCUACUUGAACAGUCU
1405
1324-1346

AD-288192.1
CAAGUAGCAUUCCAAUCUGUA
1108
1332-1352
UACAGATUGGAAUGCUACUUGAA
1408
1330-1352

AD-288227.1
AACUUAUUUACACAGGGAAGU
1111
1299-1319
ACUUCCCUGUGUAAAUAAGUUCU
1411
1297-1319

AD-288229.1
UACACAGGGAAGGUUUAAGAU
1112
1307-1327
AUCUUAAACCUUCCCUGUGUAAA
1412
1305-1327

AD-288230.1
CACAGGGAAGGUUUAAGACUU
1113
1309-1329
AAGUCUTAAACCUUCCCUGUGUA
1413
1307-1329

AD-288231.1
AGGGAAGGUUUAAGACUGUUU
1114
1312-1332
AAACAGTCUUAAACCUUCCCUGU
1414
1310-1332

AD-288232.1
GAAGGUUUAAGACUGUUCAAU
1115
1315-1335
AUUGAACAGUCUUAAACCUUCCC
1415
1313-1335

AD-288233.1
GGUUUAAGACUGUUCAAGUAU
1116
1318-1338
AUACUUGAACAGUCUUAAACCUU
1416
1316-1338

AD-288235.1
AAGACUGUUCAAGUAGCAUUU
1117
1323-1343
AAAUGCTACUUGAACAGUCUUAA
1417
1321-1343

AD-288236.1
AGACUGUUCAAGUAGCAUUCU
1118
1324-1344
AGAAUGCUACUUGAACAGUCUUA
1418
1322-1344

AD-288237.1
UGUUCAAGUAGCAUUCCAAUU
1119
1328-1348
AAUUGGAAUGCUACUUGAACAGU
1419
1326-1348

AD-288238.1
UUCAAGUAGCAUUCCAAUCUU
1120
1330-1350
AAGAUUGGAAUGCUACUUGAACA
1420
1328-1350

AD-288290.1
GCAGCUUUAUCUCAACCUGGA
1121
1419-1439
UCCAGGTUGAGAUAAAGCUGCCU
1421
1417-1439

AD-288316.1
AGCAUUUGAAAGAUUUCCCUA
1122
1455-1475
UAGGGAAAUCUUUCAAAUGCUGA
1422
1453-1475

AD-288318.1
UGAAAGAUUUCCCUAGCCUCU
1123
1461-1481
AGAGGCTAGGGAAAUCUUUCAAA
1423
1459-1481

AD-288324.1
UUCCCUAGCCUCUUCCUUUUU
1125
1469-1489
AAAAAGGAAGAGGCUAGGGAAAU
1425
1467-1489

AD-288337.1
AAGAACACAGAAUGAGUGCAU
1126
1374-1394
AUGCACTCAUUCUGUGUUCUUGU
1426
1372-1394

AD-288338.1
GAACACAGAAUGAGUGCACAU
1127
1376-1396
AUGUGCACUCAUUCUGUGUUCUU
1427
1374-1396

AD-288339.1
AACACAGAAUGAGUGCACAGU
1128
1377-1397
ACUGUGCACUCAUUCUGUGUUCU
1428
1375-1397

AD-288340.1
CAGAAUGAGUGCACAGCUAAU
1129
1381-1401
AUUAGCTGUGCACUCAUUCUGUG
1429
1379-1401

AD-288341.1
GAAUGAGUGCACAGCUAAGAU
1130
1383-1403
AUCUUAGCUGUGCACUCAUUCUG
1430
1381-1403

AD-288366.1
UUCAGCAUUUGAAAGAUUUCU
1131
1452-1472
AGAAAUCUUUCAAAUGCUGAAUC
1431
1450-1472

AD-288369.1
CAUUUGAAAGAUUUCCCUAGU
1132
1457-1477
ACUAGGGAAAUCUUUCAAAUGCU
1432
1455-1477

AD-288370.1
AUUUGAAAGAUUUCCCUAGCU
1133
1458-1478
AGCUAGGGAAAUCUUUCAAAUGC
1433
1456-1478

AD-288394.1
GUGCAACUCUAUUCUGGACUU
1136
1505-1525
AAGUCCAGAAUAGAGUUGCACCG
1436
1503-1525

AD-288396.1
GCAACUCUAUUCUGGACUUUA
1138
1507-1527
UAAAGUCCAGAAUAGAGUUGCAC
1438
1505-1527

AD-288397.1
CAACUCUAUUCUGGACUUUAU
1139
1508-1528
AUAAAGTCCAGAAUAGAGUUGCA
1439
1506-1528

AD-288427.1
AGUCCACCAAAAGUGGACCCU
1142
1559-1579
AGGGUCCACUUUUGGUGGACUUC
1442
1557-1579

AD-288466.1
AAACGGUGCAACUCUAUUCUU
1148
1500-1520
AAGAAUAGAGUUGCACCGUUUUG
1448
1498-1520

AD-288467.1
AACGGUGCAACUCUAUUCUGU
1149
1501-1521
ACAGAATAGAGUUGCACCGUUUU
1449
1499-1521

AD-288470.1
UAUUCUGGACUUUAUUACUUU
1150
1514-1534
AAAGUAAUAAAGUCCAGAAUAGA
1450
1512-1534

AD-288491.1
AAAGUGGACCCUCUAUAUUUC
1151
1568-1588
GAAAUATAGAGGGUCCACUUUUG
1451
1566-1588

AD-288492.1
AAGUGGACCCUCUAUAUUUCU
1152
1569-1589
AGAAAUAUAGAGGGUCCACUUUU
1452
1567-1589

AD-288561.1
AUGUUUAGACAAUUUUAGGCU
1153
1724-1744
AGCCUAAAAUUGUCUAAACAUCU
1453
1722-1744

AD-288562.1
GUUUAGACAAUUUUAGGCUCA
1154
1726-1746
UGAGCCTAAAAUUGUCUAAACAU
1454
1724-1746

AD-288567.1
GACAAUUUUAGGCUCAAAAAU
1159
1731-1751
AUUUUUGAGCCUAAAAUUGUCUA
1459
1729-1751

AD-288573.1
UAGGCUCAAAAAUUAAAGCUA
1161
1739-1759
UAGCUUTAAUUUUUGAGCCUAAA
1461
1737-1759

AD-288574.1
AGGCUCAAAAAUUAAAGCUAA
1162
1740-1760
UUAGCUTUAAUUUUUGAGCCUAA
1462
1738-1760

AD-288575.1
GCUCAAAAAUUAAAGCUAACA
1163
1742-1762
UGUUAGCUUUAAUUUUUGAGCCU
1463
1740-1762

AD-288576.1
UCAAAAAUUAAAGCUAACACA
1164
1744-1764
UGUGUUAGCUUUAAUUUUUGAGC
1464
1742-1764

AD-288613.1
UUCAUAUAUCCUUGGUCCCAU
1165
1701-1721
AUGGGACCAAGGAUAUAUGAAAG
1465
1699-1721

AD-288620.1
GAUGUUUAGACAAUUUUAGGU
1166
1723-1743
ACCUAAAAUUGUCUAAACAUCUC
1466
1721-1743

AD-288624.1
GGCUCAAAAAUUAAAGCUAAU
1167
1741-1761
AUUAGCTUUAAUUUUUGAGCCUA
1467
1739-1761

AD-288625.1
CUCAAAAAUUAAAGCUAACAU
1168
1743-1763
AUGUUAGCUUUAAUUUUUGAGCC
1468
1741-1763

AD-288626.1
CAAAAAUUAAAGCUAACACAU
1169
1745-1765
AUGUGUTAGCUUUAAUUUUUGAG
1469
1743-1765

AD-288628.1
AAAUUAAAGCUAACACAGGAA
1170
1748-1768
UUCCUGTGUUAGCUUUAAUUUUU
1470
1746-1768

AD-288634.1
UAACACAGGAAAAGGAACUGU
1171
1758-1778
ACAGUUCCUUUUCCUGUGUUAGC
1471
1756-1778

AD-288637.1
GGAAAAGGAACUGUACUGGCU
1172
1765-1785
AGCCAGTACAGUUCCUUUUCCUG
1472
1763-1785

AD-288696.1
CAGGAAAAGGAACUGUACUGU
1175
1763-1783
ACAGUACAGUUCCUUUUCCUGUG
1475
1761-1783

AD-288698.1
AGGAACUGUACUGGCUAUUAU
1176
1770-1790
AUAAUAGCCAGUACAGUUCCUUU
1476
1768-1790

AD-288912.1
CUCCCACUACAUCAAGACUAA
1177
2189-2209
UUAGUCTUGAUGUAGUGGGAGUC
1477
2187-2209

AD-288915.1
CACUACAUCAAGACUAAUCUU
1180
2193-2213
AAGAUUAGUCUUGAUGUAGUGGG
1480
2191-2213

AD-288916.1
CUACAUCAAGACUAAUCUUGU
1181
2195-2215
ACAAGATUAGUCUUGAUGUAGUG
1481
2193-2215

AD-288933.1
GUGUUUUUCACAUGUAUUAUA
1184
2221-2241
UAUAAUACAUGUGAAAAACACAC
1484
2219-2241

AD-288937.1
UCACAUGUAUUAUAGAAUGCU
1185
2228-2248
AGCAUUCUAUAAUACAUGUGAAA
1485
2226-2248

AD-288938.1
CACAUGUAUUAUAGAAUGCUU
1186
2229-2249
AAGCAUTCUAUAAUACAUGUGAA
1486
2227-2249

AD-288948.1
CUUUUGCAUGGACUAUCCUCU
1192
2247-2267
AGAGGATAGUCCAUGCAAAAGCA
1492
2245-2267

AD-288950.1
UUGCAUGGACUAUCCUCUUGU
1193
2250-2270
ACAAGAGGAUAGUCCAUGCAAAA
1493
2248-2270

AD-288956.1
GGACUAUCCUCUUGUUUUUAU
1196
2256-2276
AUAAAAACAAGAGGAUAGUCCAU
1496
2254-2276

AD-288957.1
GACUAUCCUCUUGUUUUUAUU
1197
2257-2277
AAUAAAAACAAGAGGAUAGUCCA
1497
2255-2277

AD-288959.1
CUAUCCUCUUGUUUUUAUUAA
1198
2259-2279
UUAAUAAAAACAAGAGGAUAGUC
1498
2257-2279

AD-288981.1
CCCACUACAUCAAGACUAAUU
1199
2191-2211
AAUUAGTCUUGAUGUAGUGGGAG
1499
2189-2211

AD-288982.1
ACUACAUCAAGACUAAUCUUU
1200
2194-2214
AAAGAUTAGUCUUGAUGUAGUGG
1500
2192-2214

AD-288996.1
AUGCUUUUGCAUGGACUAUCU
1201
2244-2264
AGAUAGTCCAUGCAAAAGCAUUC
1501
2242-2264

*Target: HSD17B13

TABLE 9

HSD17B13 ELF7 GNA7 in vitro 10 nM screen in Cos-7 (Human Dual-

Luciferase psiCHECK2 vector) and Primary Cynomolgus Hepatocytes

% of Human Message
% of Human Message
% of Cyno Message
% of Cyno Message

Duplex Name
Remaining 10 nM
Remaining 0.1 nM
remaining 10 nM
remaining 0.1 nM

AD-286958.1
108.7
111.4
84.5
99.1

AD-287001.1
84.8
98.4
14.7
81.4

AD-287012.1
88.6
110
97.1
88.8

AD-287014.1
95.2
105.8
116.5
102.3

AD-287015.1
65.4
93.8
14.5
80.3

AD-287017.1
98.5
94.9
91.6
96.2

AD-287026.1
31.9
91
44.8
73.6

AD-287059.1
76.9
117.2
70
94.4

AD-287060.1
101.6
97.9
71.1
97.8

AD-287061.1
94.6
92.8
60.5
96.4

AD-287062.1
90.8
97.7
67.1
113.1

AD-287064.1
97
92.5
75
98.2

AD-287065.1
92.2
98.2
74.4
98

AD-287066.1
98.5
96.4
27
99.3

AD-287126.1
95.6
99.9
19.1
93.5

AD-287200.1
98.7
126.7
91.5
95.3

AD-287201.1
38.7
101.6
88.6
100.6

AD-287202.1
74.5
97.2
88.7
100.7

AD-287203.1
72.9
98.7
50.3
87.1

AD-287205.1
72.8
98
40
96.9

AD-287206.1
62.7
112.9
19.6
88.4

AD-287227.1
64.3
95
34.2
95.1

AD-287246.1
58.1
97.5
20.8
72.4

AD-287247.1
76.2
95.9
84.8
115.1

AD-287248.1
65.5
110.9
55
92

AD-287249.1
90.1
107.8
126.6
113.4

AD-287250.1
76.9
113.2
96.2
95.6

AD-287251.1
35.8
92.3
53.5
93.7

AD-287252.1
57.7
89
32.7
101.4

AD-287253.1
76.1
95
74.1
92.6

AD-287254.1
104.7
124
89.5
100.5

AD-287258.1
91.4
107.8
44.9
98.1

AD-287287.1
55.2
96.5
65.7
104.4

AD-287288.1
53.1
96.9
36
108.3

AD-287289.1
77.3
98.1
57.5
97.8

AD-287290.1
23.5
103.2
55.3
82.8

AD-287293.1
46.6
89.5
88.6
102.2

AD-287316.1
78.6
82.4
34.1
92.4

AD-287320.1
86.2
88.8
105.8
97.9

AD-287321.1
86
108.2
81.3
91

AD-287324.1
72.7
113.6
34.3
107.8

AD-287325.1
78.4
104.6
85.2
86.2

AD-287326.1
39.2
95.1
17.3
73.3

AD-287327.1
91.7
95.2
99.6
90.4

AD-287344.1
13.6
81.4
14.4
58.3

AD-287346.1
115.2
122.2
96.5
103.1

AD-287347.1
43.3
89.6
26.3
86.7

AD-287366.1
53
98.4
36.1
90.1

AD-287367.1
102.8
99.3
98.4
93.3

AD-287370.1
107.7
106.7
102.1
93.8

AD-287371.1
79.2
107
81.6
93.5

AD-287372.1
101.8
103.2
94.4
108.4

AD-287373.1
67.5
85.9
28.7
89.6

AD-287374.1
60.7
103.2
63.2
83.2

AD-287375.1
96
101
85.3
98

AD-287391.1
28.3
92
26.4
95.3

AD-287392.1
33.8
100.9
31.4
90.8

AD-287406.1
93.2
106.5
54.1
96.2

AD-287407.1
47.5
86.1
23.6
72.2

AD-287408.1
63.1
90.3
47.1
98.2

AD-287410.1
32.5
87.6
10.9
80.2

AD-287411.1
21.6
87.3
10
69.1

AD-287413.1
87.4
98.2
72.7
89.9

AD-287414.1
100.6
97.3
100.2
98.6

AD-287415.1
20.2
88.9
9.7
68.8

AD-287418.1
13.3
88.8
45.9
66.6

AD-287419.1
54.6
99.7
58.7
88

AD-287421.1
27.4
94.7
7.4
66.2

AD-287424.1
33.4
98
40.6
87.6

AD-287427.1
67.9
106.6
24.2
75.8

AD-287428.1
79.1
94.5
22.3
84.7

AD-287429.1
99.5
98.2
53.8
94.7

AD-287430.1
101.9
104
88.3
103.9

AD-287441.1
62.8
104
61.1
103.6

AD-287453.1
19.9
91.5
12.5
70.3

AD-287462.1
44.1
90.3
85.1
93.1

AD-287463.1
93.9
99.9
78.3
100.4

AD-287464.1
113
107.9
51.5
110.6

AD-287465.1
86.6
99
80.4
101.5

AD-287466.1
89.9
96.4
106.2
109.7

AD-287467.1
85.2
100.4
82.2
107.9

AD-287468.1
36.6
102.8
13.7
92.4

AD-287469.1
67.5
93.6
88.5
107.8

AD-287470.1
30
111.3
24.9
68.2

AD-287474.1
61.6
103.1
55.4
93.3

AD-287476.1
33.2
120.5
29.2
52

AD-287477.1
62.3
90.6
38.2
81.3

AD-287478.1
44
86.3
6.1
52.5

AD-287479.1
89.8
95.8
53
95.1

AD-287491.1
63.1
108.1
75.7
92.2

AD-287492.1
70.8
92.9
93.6
97.1

AD-287517.1
75.2
93.3
98
99.5

AD-287520.1
73.9
97.9
96.2
101.9

AD-287521.1
50.1
94.6
70.2
98.7

AD-287522.1
88.9
109.4
83.4
91.8

AD-287524.1
14.4
111.5
31.1
65.9

AD-287525.1
71.6
104.4
88.6
103.8

AD-287537.1
51.6
110.5
20.5
84.7

AD-287539.1
47.9
115.9
27.9
78.5

AD-287540.1
74.9
105.1
19.6
86.2

AD-287543.1
47.6
95.3
12.7
93.3

AD-287546.1
65.7
102.8
6.8
59

AD-287568.1
74.8
104.6
56.7
100.7

AD-287591.1
40
100.8
12.7
65.1

AD-287610.1
33.3
78.1
32.3
66.4

AD-287615.1
24.4
104.8
12.5
68.7

AD-287616.1
15.7
102
16.1
63.6

AD-287627.1
81.3
92.3
111.8
94.5

AD-287632.1
93.9
93.1
88.9
106.9

AD-287633.1
106.4
91.9
68.4
122.6

AD-287634.1
82.3
114.7
78.7
94.5

AD-287636.1
21.8
97.7
18.9
56.5

AD-287637.1
27.6
92.3
22.8
91.4

AD-287638.1
99
101.2
101.6
98.1

AD-287647.1
74.2
108.2
74.5
94.3

AD-287648.1
12.9
92.8
29.8
61

AD-287651.1
66.5
100.6
92.8
106.4

AD-287652.1
12.8
79.5
11.5
66.2

AD-287653.1
18.9
69.8
17.9
87.7

AD-287654.1
87.5
101
75.4
91.4

AD-287655.1
61.6
101.7
88.3
98

AD-287656.1
91.7
122.5
86.4
97.9

AD-287658.1
78.1
118.7
45
89.4

AD-287659.1
37
100.9
22.1
87.2

AD-287679.1
55.4
93.6
24.4
85.1

AD-287680.1
14.3
114.1
17.4
35.8

AD-287683.1
59
94.1
33.3
87.5

AD-287690.1
81.2
144.4
49.9
93.5

AD-287693.1
68.7
125.5
85.8
100.5

AD-287694.1
100.2
111.7
87.8
101.8

AD-287695.1
88.4
110.2
89.2
106.1

AD-287696.1
13.8
78.3
12
63.6

AD-287700.1
15.6
97
22.9
82.3

AD-287701.1
46.1
95
108.7
109.1

AD-287702.1
80.1
112.2
80.1
93.9

AD-287703.1
70.1
102.6
92.2
105.5

AD-287704.1
38.2
113.8
25.4
95.5

AD-287706.1
94.3
105
96.1
97.8

AD-287707.1
82.6
114.2
103.2
106.7

AD-287709.1
78.2
93.8
89.9
103.9

AD-287711.1
23.1
76.9
15.5
88.1

AD-287724.1
54.2
94.9
100.4
97.6

AD-287725.1
83.1
116.3
61.8
101.3

AD-287726.1
20.8
77.6
15.5
70.6

AD-287727.1
33.6
111
27.9
57.1

AD-287728.1
28.8
107
11.2
76.6

AD-287735.1
13.2
92.7
9.6
25.6

AD-287736.1
60.7
111
26.9
83.8

AD-287737.1
71.3
98.7
42.5
105.1

AD-287738.1
31.3
114.3
21.8
87.8

AD-287739.1
22.5
101.9
55
72

AD-287740.1
16.3
73.5
19
55.7

AD-287778.1
82.4
113.7
66.4
90.9

AD-287793.1
47.8
91.5
84.1
92.9

AD-287795.1
32.9
99.6
54.4
102.2

AD-287798.1
48.8
98.3
18.5
90.5

AD-287800.1
59.4
96.3
58.7
95.1

AD-287801.1
17.7
140.3
16.1
71.4

AD-287802.1
5.6
82.9
17
27.8

AD-287804.1
14.7
77.5
14.3
70.3

AD-287810.1
53.9
98.6
72.5
100.1

AD-287811.1
64.5
106
33.9
90.2

AD-287813.1
35.9
114.4
17.9
91.4

AD-287837.1
11.6
66
9.3
58

AD-287839.1
95.3
106.1
60
136.1

AD-287841.1
13.4
98.7
22.9
46.4

AD-287842.1
94.6
89.9
106.1
97.5

AD-287844.1
87.4
161.1
74
88.1

AD-287854.1
32.4
103.1
25.2
82.8

AD-287856.1
16.9
99.3
32.4
64.5

AD-287857.1
88
99.4
64.7
106.2

AD-287893.1
17.2
94.7
58.7
93.2

AD-287917.1
30.5
114.1
60
69.4

AD-287924.1
52.3
85.6
75.3
86.5

AD-287934.1
48.5
91.3
39.4
96.8

AD-287969.1
100.3
96.7
77.9
105.5

AD-287970.1
15.4
88.3
43
87.6

AD-287982.1
73.8
87.6
26.7
56.3

AD-288005.1
23.6
92.4
69.9
88.4

AD-288006.1
28.8
91.1
52.8
95.3

AD-288008.1
62.6
111.1
52
97.5

AD-288010.1
66.8
116.2
66.1
102

AD-288011.1
11.7
78
45.2
54.5

AD-288015.1
27.1
111.4
66.6
61

AD-288046.1
12.2
72.4
20.7
53.5

AD-288049.1
44.9
104.5
68.6
77.3

AD-288050.1
32.2
120.1
41.8
70.6

AD-288076.1
43.1
109.1
24.2
66.4

AD-288092.1
10.9
106.6
58.5
113.2

AD-288094.1
66.8
92.3
90.4
91.6

AD-288095.1
16.2
92.4
39
55.5

AD-288108.1
53.3
122.8
49.1
96.7

AD-288109.1
39.8
114.6
51
90.4

AD-288173.1
19.3
110.7
58.8
81.1

AD-288174.1
39
90.1
71.8
88.7

AD-288176.1
135.9
92.9
102.6
106

AD-288177.1
91.4
99.7
57.2
99.5

AD-288180.1
34.5
110
68
82

AD-288181.1
19.9
116.4
34.6
74.9

AD-288183.1
38.2
96.5
20.5
84.6

AD-288184.1
43.4
95
28.9
84.1

AD-288186.1
63.9
107.4
60.7
88

AD-288187.1
22.9
113.8
53
84.5

AD-288188.1
16.3
77.6
23.4
60.5

AD-288189.1
47.2
104.1
22.4
49.3

AD-288190.1
21.5
81.3
22.8
58.1

AD-288192.1
71.7
112.4
76.6
93.6

AD-288193.1
62.5
118.5
34
72.8

AD-288194.1
66.7
96.6
31.2
87.1

AD-288227.1
86.5
85.7
113.1
103.6

AD-288229.1
104.6
103.1
73.7
88.6

AD-288230.1
32.5
122.3
29.7
75.1

AD-288231.1
19
100.2
46.5
72.1

AD-288232.1
66.9
95.5
97
95.2

AD-288233.1
26.2
108
21.1
68.6

AD-288235.1
7.4
79.8
39.3
52.6

AD-288236.1
18.1
78.8
21.5
51.6

AD-288237.1
59.8
133.8
37.3
72.6

AD-288238.1
10.4
57.4
21.3
58.1

AD-288290.1
68.9
108.4
29.8
90.7

AD-288316.1
38
98.2
42.2
61.8

AD-288318.1
37.6
99.4
25.3
66

AD-288319.1
23.7
93.5
44.4
90.4

AD-288324.1
56.4
146.7
51.7
81.8

AD-288337.1
24.7
94.5
30
78

AD-288338.1
34.8
95.6
46.9
68.3

AD-288339.1
67.3
86.5
78.5
94

AD-288340.1
19.5
114.6
29.5
51.7

AD-288341.1
23.1
88.9
48.8
75.7

AD-288366.1
34.6
160.9
42.9
91.4

AD-288369.1
16.4
120.9
28.2
77.4

AD-288370.1
15.1
73.8
26.8
89.1

AD-288391.1
30.1
84
21.2
64.5

AD-288393.1
56
96.6
41.8
101.9

AD-288394.1
20.1
107
48.5
75.6

AD-288395.1
64
94.8
53.7
101

AD-288396.1
86
94.3
100.2
90.7

AD-288397.1
28.3
83.9
33.9
83.9

AD-288398.1
63.4
108.8
87.6
91.7

AD-288402.1
60.1
103.7
37
89.3

AD-288427.1
110.8
95.6
89.8
97.2

AD-288430.1
94.7
118.3
85.2
92.7

AD-288431.1
87.4
94.7
106.5
94.5

AD-288432.1
39
112.5
81.6
88.7

AD-288433.1
100.7
113
107.9
92.4

AD-288434.1
104.8
99.6
87.5
102.3

AD-288466.1
65
99.1
77.1
100

AD-288467.1
99.4
99.8
64.9
98.6

AD-288470.1
30.7
97.7
82.9
83.8

AD-288491.1
82.6
98.5
69.8
90.6

AD-288492.1
77
98.1
84.5
106.6

AD-288561.1
54.5
92.2
102.4
103.6

AD-288562.1
35.9
91.8
32.2
89.5

AD-288563.1
18.7
79.9
29.9
69.1

AD-288564.1
24.5
68.1
38.3
80.9

AD-288565.1
47.1
97.6
53.9
83.8

AD-288566.1
33.7
89.8
30.5
64.6

AD-288567.1
75.7
92.5
101.7
99.7

AD-288572.1
93.2
97.6
92
110.6

AD-288573.1
51.9
95
46.3
86.1

AD-288574.1
12.6
79.8
25.8
54.3

AD-288575.1
51.2
116.2
61.7
76.2

AD-288576.1
26.9
98
51.7
75

AD-288613.1
36
92.1
103.9
113.2

AD-288620.1
62.8
94.8
107
88.5

AD-288624.1
26.8
101.4
32.2
66.8

AD-288625.1
16.4
99.3
40.5
73.2

AD-288626.1
34.7
100.9
30.1
70.8

AD-288628.1
32.7
85.2
35.1
80.2

AD-288634.1
89.6
92.6
77
90.6

AD-288637.1
44.5
93.6
42.1
104

AD-288639.1
43.7
112
20.6
46.7

AD-288642.1
42.2
99.6
25.2
69.8

AD-288696.1
52.8
101.5
31.1
88

AD-288698.1
16.3
75.6
21
58.9

AD-288912.1
21.1
83.4
23.2
56.1

AD-288913.1
28.5
88
26.7
65.9

AD-288914.1
16.1
77.7
23.1
56.1

AD-288915.1
17.7
92.8
33.9
73.6

AD-288916.1
16.1
91.1
24
75.5

AD-288917.1
15.1
59.6
31
77

AD-288918.1
23.6
85.2
35.2
98.8

AD-288933.1
24.9
78.9
62.9
78.8

AD-288937.1
74.3
103.3
51.6
89.9

AD-288938.1
28.2
105.7
42.9
92.7

AD-288941.1
33.1
100
44.9
114.1

AD-288943.1
69.7
97.2
49.1
89

AD-288945.1
25.1
83.4
26.1
64.4

AD-288946.1
54
89.5
45.5
82.9

AD-288947.1
26.7
97.6
26.6
76.9

AD-288948.1
18.2
93.6
24.7
67.6

AD-288950.1
24.3
103.4
31.8
89.7

AD-288951.1
18.9
85.3
29.7
96.5

AD-288952.1
98.1
103.2
85.4
92.9

AD-288956.1
30.5
104
53
84.5

AD-288957.1
23.4
88.8
57.9
85.2

AD-288959.1
21.5
82.5
45.7
64.4

AD-288981.1
63.2
101.9
26.6
73.5

AD-288982.1
82.9
103.7
50.2
88.4

AD-288996.1
11.6
70.8
39.3
70.3

TABLE 10

HSD17B13 NonF DNA Modified Sequence*

mRNA

Duplex Name
Sense sequence 5′ to 3′
SEQ ID NO:
Antisense sequence 5′ to 3′
SEQ ID NO:
target sequence 5′ to 3′
SEQ ID NO:

AD-291929.1
ascsacaaggdAcdTgaaccagaauL96
1502
asdTsucdTgdGuucadGudCcuugugusasg
1797
CUACACAAGGACUGAACCAGAAG
2092

AD-291931.1
ascsaaggacdTgdAaccagaaggaL96
1503
usdCscudTcdTgguudCadGuccuugusgsu
1798
ACACAAGGACUGAACCAGAAGGA
2093

AD-291988.1
uscscuucugdCudTcugaucaccaL96
1504
usdGsgudGadTcagadAgdCagaaggasusu
1799
AAUCCUUCUGCUUCUGAUCACCA
2094

AD-291993.1
csusgcuucudGadTcaccaucauuL96
1505
asdAsugdAudGgugadTcdAgaagcagsasa
1800
UUCUGCUUCUGAUCACCAUCAUC
2095

AD-291998.1
uscsugaucadCcdAucaucuacuuL96
1506
asdAsgudAgdAugaudGgdTgaucagasasg
1801
CUUCUGAUCACCAUCAUCUACUC
2096

AD-292005.1
ascscaucaudCudAcuccuacuuuL96
1507
asdAsagdTadGgagudAgdAugauggusgsa
1802
UCACCAUCAUCUACUCCUACUUG
2097

AD-292006.1
cscsaucaucdTadCuccuacuuguL96
1508
asdCsaadGudAggagdTadGaugauggsusg
1803
CACCAUCAUCUACUCCUACUUGG
2098

AD-292007.1
csasucaucudAcdTccuacuuggaL96
1509
usdCscadAgdTaggadGudAgaugaugsgsu
1804
ACCAUCAUCUACUCCUACUUGGA
2099

AD-292008.1
asuscaucuadCudCcuacuuggauL96
1510
asdTsccdAadGuaggdAgdTagaugausgsg
1805
CCAUCAUCUACUCCUACUUGGAG
2100

AD-292010.1
csasucuacudCcdTacuuggaguuL96
1511
asdAscudCcdAaguadGgdAguagaugsasu
1806
AUCAUCUACUCCUACUUGGAGUC
2101

AD-292012.1
uscsuacuccdTadCuuggagucguL96
1512
asdCsgadCudCcaagdTadGgaguagasusg
1807
CAUCUACUCCUACUUGGAGUCGU
2102

AD-292013.1
csusacuccudAcdTuggagucguuL96
1513
asdAscgdAcdTccaadGudAggaguagsasu
1808
AUCUACUCCUACUUGGAGUCGUU
2103

AD-292014.1
usascuccuadCudTggagucguuuL96
1514
asdAsacdGadCuccadAgdTaggaguasgsa
1809
UCUACUCCUACUUGGAGUCGUUG
2104

AD-292015.1
ascsuccuacdTudGgagucguuguL96
1515
asdCsaadCgdAcuccdAadGuaggagusasg
1810
CUACUCCUACUUGGAGUCGUUGG
2105

AD-292016.1
csusccuacudTgdGagucguugguL96
1516
asdCscadAcdGacucdCadAguaggagsusa
1811
UACUCCUACUUGGAGUCGUUGGU
2106

AD-292017.1
uscscuacuudGgdAgucguugguuL96
1517
asdAsccdAadCgacudCcdAaguaggasgsu
1812
ACUCCUACUUGGAGUCGUUGGUG
2107

AD-292028.1
gsuscguuggdTgdAaguuuuucauL96
1518
asdTsgadAadAacuudCadCcaacgacsusc
1813
GAGUCGUUGGUGAAGUUUUUCAU
2108

AD-292137.1
ususgguucudGudGggauauuaauL96
1519
asdTsuadAudAucccdAcdAgaaccaasusa
1814
UAUUGGUUCUGUGGGAUAUUAAU
2109

AD-292140.1
gsusucugugdGgdAuauuaauaauL96
1520
asdTsuadTudAauaudCcdCacagaacscsa
1815
UGGUUCUGUGGGAUAUUAAUAAG
2110

AD-292143.1
csusgugggadTadTuaauaageguL96
1521
asdCsgcdTudAuuaadTadTcccacagsasa
1816
UUCUGUGGGAUAUUAAUAAGCGC
2111

AD-292182.1
gscsugagugdCcdGaaaacuagguL96
1522
asdCscudAgdTuuucdGgdCacucagcsusg
1817
CAGCUGAGUGCCGAAAACUAGGC
2112

AD-292183.1
csusgagugcdCgdAaaacuaggcuL96
1523
asdGsccdTadGuuuudCgdGcacucagscsu
1818
AGCUGAGUGCCGAAAACUAGGCG
2113

AD-292185.1
gsasgugccgdAadAacuaggcguuL96
1524
asdAscgdCcdTaguudTudCggcacucsasg
1819
CUGAGUGCCGAAAACUAGGCGUC
2114

AD-292186.1
asgsugccgadAadAcuaggcgucaL96
1525
usdGsacdGcdCuagudTudTcggcacuscsa
1820
UGAGUGCCGAAAACUAGGCGUCA
2115

AD-292187.1
gsusgccgaadAadCuaggcgucauL96
1526
asdTsgadCgdCcuagdTudTucggcacsusc
1821
GAGUGCCGAAAACUAGGCGUCAC
2116

AD-292188.1
usgsccgaaadAcdTaggcgucacuL96
1527
asdGsugdAcdGccuadGudTuucggcascsu
1822
AGUGCCGAAAACUAGGCGUCACU
2117

AD-292189.1
gscscgaaaadCudAggcgucacuuL96
1528
asdAsgudGadCgccudAgdTuuucggcsasc
1823
GUGCCGAAAACUAGGCGUCACUG
2118

AD-292190.1
cscsgaaaacdTadGgcgucacuguL96
1529
asdCsagdTgdAcgccdTadGuuuucggscsa
1824
UGCCGAAAACUAGGCGUCACUGC
2119

AD-292238.1
csasgagaagdAgdAucuaucgcuuL96
1530
asdAsgedGadTagaudCudCuucucugsusu
1825
AACAGAGAAGAGAUCUAUCGCUC
2120

AD-292244.1
asgsagaucudAudCgcucucuaaaL96
1531
usdTsuadGadGagcgdAudAgaucucususc
1826
GAAGAGAUCUAUCGCUCUCUAAA
2121

AD-292245.1
gsasgaucuadTcdGcucucuaaauL96
1532
asdTsuudAgdAgagedGadTagaucucsusu
1827
AAGAGAUCUAUCGCUCUCUAAAU
2122

AD-292247.1
gsasucuaucdGcdTcucuaaaucaL96
1533
usdGsaudTudAgagadGcdGauagaucsusc
1828
GAGAUCUAUCGCUCUCUAAAUCA
2123

AD-292248.1
asuscuaucgdCudCucuaaaucauL96
1534
asdTsgadTudTagagdAgdCgauagauscsu
1829
AGAUCUAUCGCUCUCUAAAUCAG
2124

AD-292250.1
csusaucgcudCudCuaaaucagguL96
1535
asdCscudGadTuuagdAgdAgcgauagsasu
1830
AUCUAUCGCUCUCUAAAUCAGGU
2125

AD-292251.1
usasucgcucdTcdTaaaucagguuL96
1536
asdAsccdTgdAuuuadGadGagcgauasgsa
1831
UCUAUCGCUCUCUAAAUCAGGUG
2126

AD-292252.1
asuscgcucudCudAaaucaggugaL96
1537
usdCsacdCudGauuudAgdAgagcgausasg
1832
CUAUCGCUCUCUAAAUCAGGUGA
2127

AD-292253.1
uscsgcucucdTadAaucaggugaaL96
1538
usdTscadCcdTgauudTadGagagcgasusa
1833
UAUCGCUCUCUAAAUCAGGUGAA
2128

AD-292254.1
csgscucucudAadAucaggugaauL96
1539
asdTsucdAcdCugaudTudAgagagcgsasu
1834
AUCGCUCUCUAAAUCAGGUGAAG
2129

AD-292257.1
uscsucuaaadTcdAggugaagaaaL96
1540
usdTsucdTudCaccudGadTuuagagasgsc
1835
GCUCUCUAAAUCAGGUGAAGAAA
2130

AD-292309.1
asusgcugggdAcdAguauauccauL96
1541
asdTsggdAudAuacudGudCccagcaususa
1836
UAAUGCUGGGACAGUAUAUCCAG
2131

AD-292310.1
usgscugggadCadGuauauccaguL96
1542
asdCsugdGadTauacdTgdTcccagcasusu
1837
AAUGCUGGGACAGUAUAUCCAGC
2132

AD-292315.1
gsgsacaguadTadTccagccgauuL96
1543
asdAsucdGgdCuggadTadTacuguccscsa
1838
UGGGACAGUAUAUCCAGCCGAUC
2133

AD-292318.1
csasguauaudCcdAgccgaucuuuL96
1544
asdAsagdAudCggcudGgdAuauacugsusc
1839
GACAGUAUAUCCAGCCGAUCUUC
2134

AD-292344.1
ascscaaggadTgdAagagauuacuL96
1545
asdGsuadAudCucuudCadTccuuggusgsc
1840
GCACCAAGGAUGAAGAGAUUACC
2135

AD-292345.1
cscsaaggaudGadAgagauuaccaL96
1546
usdGsgudAadTcucudTcdAuccuuggsusg
1841
CACCAAGGAUGAAGAGAUUACCA
2136

AD-292346.1
csasaggaugdAadGagauuaccaaL96
1547
usdTsggdTadAucucdTudCauccuugsgsu
1842
ACCAAGGAUGAAGAGAUUACCAA
2137

AD-292363.1
cscsaagacadTudTgaggucaacaL96
1548
usdGsuudGadCcucadAadTgucuuggsusa
1843
UACCAAGACAUUUGAGGUCAACA
2138

AD-292366.1
asgsacauuudGadGgucaacaucuL96
1549
asdGsaudGudTgaccdTcdAaaugucususg
1844
CAAGACAUUUGAGGUCAACAUCC
2139

AD-292367.1
gsascauuugdAgdGucaacauccuL96
1550
asdGsgadTgdTugacdCudCaaaugucsusu
1845
AAGACAUUUGAGGUCAACAUCCU
2140

AD-292369.1
csasuuugagdGudCaacauccuauL96
1551
asdTsagdGadTguugdAcdCucaaaugsusc
1846
GACAUUUGAGGUCAACAUCCUAG
2141

AD-292370.1
asusuugaggdTcdAacauccuaguL96
1552
asdCsuadGgdAuguudGadCcucaaausgsu
1847
ACAUUUGAGGUCAACAUCCUAGG
2142

AD-292371.1
ususugaggudCadAcauccuaggaL96
1553
usdCscudAgdGaugudTgdAccucaaasusg
1848
CAUUUGAGGUCAACAUCCUAGGA
2143

AD-292373.1
usgsaggucadAcdAuccuaggacaL96
1554
usdGsucdCudAggaudGudTgaccucasasa
1849
UUUGAGGUCAACAUCCUAGGACA
2144

AD-292374.1
gsasggucaadCadTccuaggacauL96
1555
asdTsgudCcdTaggadTgdTugaccucsasa
1850
UUGAGGUCAACAUCCUAGGACAU
2145

AD-292376.1
gsgsucaacadTcdCuaggacauuuL96
1556
asdAsaudGudCcuagdGadTguugaccsusc
1851
GAGGUCAACAUCCUAGGACAUUU
2146

AD-292377.1
gsuscaacaudCcdTaggacauuuuL96
1557
asdAsaadTgdTccuadGgdAuguugacscsu
1852
AGGUCAACAUCCUAGGACAUUUU
2147

AD-292379.1
csasacauccdTadGgacauuuuugL96
1558
csdAsaadAadTguccdTadGgauguugsasc
1853
GUCAACAUCCUAGGACAUUUUUG
2148

AD-292380.1
asascauccudAgdGacauuuuuguL96
1559
asdCsaadAadAugucdCudAggauguusgsa
1854
UCAACAUCCUAGGACAUUUUUGG
2149

AD-292381.1
ascsauccuadGgdAcauuuuuggaL96
1560
usdCscadAadAaugudCcdTaggaugususg
1855
CAACAUCCUAGGACAUUUUUGGA
2150

AD-292382.1
csasuccuagdGadCauuuuuggauL96
1561
asdTsccdAadAaaugdTcdCuaggaugsusu
1856
AACAUCCUAGGACAUUUUUGGAU
2151

AD-292383.1
asusccuaggdAcdAuuuuuggauuL96
1562
asdAsucdCadAaaaudGudCcuaggausgsu
1857
ACAUCCUAGGACAUUUUUGGAUC
2152

AD-292384.1
uscscuaggadCadTuuuuggaucaL96
1563
usdGsaudCcdAaaaadTgdTccuaggasusg
1858
CAUCCUAGGACAUUUUUGGAUCA
2153

AD-292385.1
cscsuaggacdAudTuuuggaucauL96
1564
asdTsgadTcdCaaaadAudGuccuaggsasu
1859
AUCCUAGGACAUUUUUGGAUCAC
2154

AD-292388.1
asgsgacauudTudTggaucacaaaL96
1565
usdTsugdTgdAuccadAadAauguccusasg
1860
CUAGGACAUUUUUGGAUCACAAA
2155

AD-292389.1
gsgsacauuudTudGgaucacaaaaL96
1566
usdTsuudGudGauccdAadAaauguccsusa
1861
UAGGACAUUUUUGGAUCACAAAA
2156

AD-292390.1
gsascauuuudTgdGaucacaaaauL96
1567
asdTsuudTgdTgaucdCadAaaaugucscsu
1862
AGGACAUUUUUGGAUCACAAAAG
2157

AD-292394.1
ususuuuggadTcdAcaaaagcacuL96
1568
asdGsugdCudTuugudGadTccaaaaasusg
1863
CAUUUUUGGAUCACAAAAGCACU
2158

AD-292397.1
ususggaucadCadAaagcacuucuL96
1569
asdGsaadGudGcuuudTgdTgauccaasasa
1864
UUUUGGAUCACAAAAGCACUUCU
2159

AD-292398.1
usgsgaucacdAadAagcacuucuuL96
1570
asdAsgadAgdTgcuudTudGugauccasasa
1865
UUUGGAUCACAAAAGCACUUCUU
2160

AD-292399.1
gsgsaucacadAadAgcacuucuuuL96
1571
asdAsagdAadGugcudTudTgugauccsasa
1866
UUGGAUCACAAAAGCACUUCUUC
2161

AD-292403.1
csascaaaagdCadCuucuuccauuL96
1572
asdAsugdGadAgaagdTgdCuuuugugsasu
1867
AUCACAAAAGCACUUCUUCCAUC
2162

AD-292404.1
ascsaaaagcdAcdTucuuccaucuL96
1573
asdGsaudGgdAagaadGudGcuuuugusgsa
1868
UCACAAAAGCACUUCUUCCAUCG
2163

AD-292405.1
csasaaagcadCudTcuuccaucgaL96
1574
usdCsgadTgdGaagadAgdTgcuuuugsusg
1869
CACAAAAGCACUUCUUCCAUCGA
2164

AD-292406.1
asasaagcacdTudCuuccaucgauL96
1575
asdTscgdAudGgaagdAadGugcuuuusgsu
1870
ACAAAAGCACUUCUUCCAUCGAU
2165

AD-292407.1
asasagcacudTcdTuccaucgauuL96
1576
asdAsucdGadTggaadGadAgugcuuususg
1871
CAAAAGCACUUCUUCCAUCGAUG
2166

AD-292408.1
asasgcacuudCudTccaucgaugaL96
1577
usdCsaudCgdAuggadAgdAagugcuususu
1872
AAAAGCACUUCUUCCAUCGAUGA
2167

AD-292409.1
asgscacuucdTudCcaucgaugauL96
1578
asdTscadTcdGauggdAadGaagugcususu
1873
AAAGCACUUCUUCCAUCGAUGAU
2168

AD-292410.1
gscsacuucudTcdCaucgaugauuL96
1579
asdAsucdAudCgaugdGadAgaagugcsusu
1874
AAGCACUUCUUCCAUCGAUGAUG
2169

AD-292434.1
asgsaaaucadTgdGccacaucguuL96
1580
asdAscgdAudGuggcdCadTgauuucuscsu
1875
AGAGAAAUCAUGGCCACAUCGUC
2170

AD-292486.1
ususccuuacdCudCaucccauauuL96
1581
asdAsuadTgdGgaugdAgdGuaaggaasusc
1876
GAUUCCUUACCUCAUCCCAUAUU
2171

AD-292487.1
uscscuuaccdTcdAucccauauuuL96
1582
asdAsaudAudGggaudGadGguaaggasasu
1877
AUUCCUUACCUCAUCCCAUAUUG
2172

AD-292492.1
ascscucaucdCcdAuauuguuccaL96
1583
usdGsgadAcdAauaudGgdGaugaggusasa
1878
UUACCUCAUCCCAUAUUGUUCCA
2173

AD-292495.1
uscsaucccadTadTuguuccagcaL96
1584
usdGscudGgdAacaadTadTgggaugasgsg
1879
CCUCAUCCCAUAUUGUUCCAGCA
2174

AD-292498.1
uscsccauaudTgdTuccagcaaauL96
1585
asdTsuudGcdTggaadCadAuaugggasusg
1880
CAUCCCAUAUUGUUCCAGCAAAU
2175

AD-292499.1
cscscauauudGudTccagcaaauuL96
1586
asdAsuudTgdCuggadAcdAauaugggsasu
1881
AUCCCAUAUUGUUCCAGCAAAUU
2176

AD-292526.1
usgsuuggcudTudCacagaggucuL96
1587
asdGsacdCudCugugdAadAgccaacasgsc
1882
GCUGUUGGCUUUCACAGAGGUCU
2177

AD-292531.1
gscsuuucacdAgdAggucugacauL96
1588
asdTsgudCadGaccudCudGugaaagcscsa
1883
UGGCUUUCACAGAGGUCUGACAU
2178

AD-292534.1
ususcacagadGgdTcugacaucauL96
1589
asdTsgadTgdTcagadCcdTcugugaasasg
1884
CUUUCACAGAGGUCUGACAUCAG
2179

AD-292536.1
csascagaggdTcdTgacaucagaaL96
1590
usdTscudGadTgucadGadCcucugugsasa
1885
UUCACAGAGGUCUGACAUCAGAA
2180

AD-292541.1
asgsgucugadCadTcagaacuucaL96
1591
usdGsaadGudTcugadTgdTcagaccuscsu
1886
AGAGGUCUGACAUCAGAACUUCA
2181

AD-292566.1
ususgggaaadAadCugguaucaaaL96
1592
usdTsugdAudAccagdTudTuucccaasgsg
1887
CCUUGGGAAAAACUGGUAUCAAA
2182

AD-292574.1
asascugguadTcdAaaaccucauuL96
1593
asdAsugdAgdGuuuudGadTaccaguususu
1888
AAAACUGGUAUCAAAACCUCAUG
2183

AD-292575.1
ascsugguaudCadAaaccucauguL96
1594
asdCsaudGadGguuudTgdAuaccagususu
1889
AAACUGGUAUCAAAACCUCAUGU
2184

AD-292576.1
csusgguaucdAadAaccucauguuL96
1595
asdAscadTgdAgguudTudGauaccagsusu
1890
AACUGGUAUCAAAACCUCAUGUC
2185

AD-292577.1
usgsguaucadAadAccucaugucuL96
1596
asdGsacdAudGaggudTudTgauaccasgsu
1891
ACUGGUAUCAAAACCUCAUGUCU
2186

AD-292578.1
gsgsuaucaadAadCcucaugucuuL96
1597
asdAsgadCadTgaggdTudTugauaccsasg
1892
CUGGUAUCAAAACCUCAUGUCUC
2187

AD-292579.1
gsusaucaaadAcdCucaugucucuL96
1598
asdGsagdAcdAugagdGudTuugauacscsa
1893
UGGUAUCAAAACCUCAUGUCUCU
2188

AD-292582.1
uscsaaaaccdTcdAugucucugcuL96
1599
asdGscadGadGacaudGadGguuuugasusa
1894
UAUCAAAACCUCAUGUCUCUGCC
2189

AD-292596.1
csuscugcccdAgdTuuuugugaauL96
1600
asdTsucdAcdAaaaadCudGggcagagsasc
1895
GUCUCUGCCCAGUUUUUGUGAAU
2190

AD-292601.1
cscscaguuudTudGugaauacuguL96
1601
asdCsagdTadTucacdAadAaacugggscsa
1896
UGCCCAGUUUUUGUGAAUACUGG
2191

AD-292606.1
ususuuugugdAadTacuggguucaL96
1602
usdGsaadCcdCaguadTudCacaaaaascsu
1897
AGUUUUUGUGAAUACUGGGUUCA
2192

AD-292607.1
ususuugugadAudAcuggguucauL96
1603
asdTsgadAcdCcagudAudTcacaaaasasc
1898
GUUUUUGUGAAUACUGGGUUCAC
2193

AD-292610.1
usgsugaauadCudGgguucaccaaL96
1604
usdTsggdTgdAacccdAgdTauucacasasa
1899
UUUGUGAAUACUGGGUUCACCAA
2194

AD-292612.1
usgsaauacudGgdGuucaccaaaaL96
1605
usdTsuudGgdTgaacdCcdAguauucascsa
1900
UGUGAAUACUGGGUUCACCAAAA
2195

AD-292613.1
gsasauacugdGgdTucaccaaaaaL96
1606
usdTsuudTgdGugaadCcdCaguauucsasc
1901
GUGAAUACUGGGUUCACCAAAAA
2196

AD-292614.1
asasuacuggdGudTcaccaaaaauL96
1607
asdTsuudTudGgugadAcdCcaguauuscsa
1902
UGAAUACUGGGUUCACCAAAAAU
2197

AD-292615.1
asusacugggdTudCaccaaaaauuL96
1608
asdAsuudTudTggugdAadCccaguaususc
1903
GAAUACUGGGUUCACCAAAAAUC
2198

AD-292616.1
usascugggudTcdAccaaaaaucuL96
1609
asdGsaudTudTuggudGadAcccaguasusu
1904
AAUACUGGGUUCACCAAAAAUCC
2199

AD-292624.1
uscsaccaaadAadTccaagcacaaL96
1610
usdTsgudGcdTuggadTudTuuggugasasc
1905
GUUCACCAAAAAUCCAAGCACAA
2200

AD-292625.1
csasccaaaadAudCcaagcacaauL96
1611
asdTsugdTgdCuuggdAudTuuuggugsasa
1906
UUCACCAAAAAUCCAAGCACAAG
2201

AD-292629.1
asasaaauccdAadGcacaagauuaL96
1612
usdAsaudCudTgugcdTudGgauuuuusgsg
1907
CCAAAAAUCCAAGCACAAGAUUA
2202

AD-292630.1
asasaauccadAgdCacaagauuauL96
1613
asdTsaadTcdTugugdCudTggauuuususg
1908
CAAAAAUCCAAGCACAAGAUUAU
2203

AD-292631.1
asasauccaadGcdAcaagauuauuL96
1614
asdAsuadAudCuugudGcdTuggauuususu
1909
AAAAAUCCAAGCACAAGAUUAUG
2204

AD-292634.1
uscscaagcadCadAgauuauggcuL96
1615
asdGsccdAudAaucudTgdTgcuuggasusu
1910
AAUCCAAGCACAAGAUUAUGGCC
2205

AD-292638.1
asgscacaagdAudTauggccuguaL96
1616
usdAscadGgdCcauadAudCuugugcususg
1911
CAAGCACAAGAUUAUGGCCUGUA
2206

AD-292639.1
gscsacaagadTudAuggccuguauL96
1617
asdTsacdAgdGccaudAadTcuugugcsusu
1912
AAGCACAAGAUUAUGGCCUGUAU
2207

AD-292640.1
csascaagaudTadTggccuguauuL96
1618
asdAsuadCadGgccadTadAucuugugscsu
1913
AGCACAAGAUUAUGGCCUGUAUU
2208

AD-292641.1
ascsaagauudAudGgccuguauuuL96
1619
asdAsaudAcdAggccdAudAaucuugusgsc
1914
GCACAAGAUUAUGGCCUGUAUUG
2209

AD-292642.1
csasagauuadTgdGccuguauuguL96
1620
asdCsaadTadCaggcdCadTaaucuugsusg
1915
CACAAGAUUAUGGCCUGUAUUGG
2210

AD-292646.1
asusuauggcdCudGuauuggagauL96
1621
asdTscudCcdAauacdAgdGccauaauscsu
1916
AGAUUAUGGCCUGUAUUGGAGAC
2211

AD-292650.1
usgsgccugudAudTggagacagauL96
1622
asdTscudGudCuccadAudAcaggccasusa
1917
UAUGGCCUGUAUUGGAGACAGAU
2212

AD-292651.1
gsgsccuguadTudGgagacagauuL96
1623
asdAsucdTgdTcuccdAadTacaggccsasu
1918
AUGGCCUGUAUUGGAGACAGAUG
2213

AD-292652.1
gscscuguaudTgdGagacagaugaL96
1624
usdCsaudCudGucucdCadAuacaggcscsa
1919
UGGCCUGUAUUGGAGACAGAUGA
2214

AD-292667.1
asgsaugaagdTcdGuaagaagucuL96
1625
asdGsacdTudCuuacdGadCuucaucusgsu
1920
ACAGAUGAAGUCGUAAGAAGUCU
2215

AD-292668.1
gsasugaagudCgdTaagaagucuuL96
1626
asdAsgadCudTcuuadCgdAcuucaucsusg
1921
CAGAUGAAGUCGUAAGAAGUCUG
2216

AD-292671.1
gsasague gudAadGaagucugauaL96
1627
usdAsucdAgdAcuucdTudAcgacuucsasu
1922
AUGAAGUCGUAAGAAGUCUGAUA
2217

AD-292673.1
asgsucguaadGadAgucugauagaL96
1628
usdCsuadTcdAgacudTcdTuacgacususc
1923
GAAGUCGUAAGAAGUCUGAUAGA
2218

AD-292677.1
gsusaagaagdTcdTgauagauggaL96
1629
usdCscadTcdTaucadGadCuucuuacsgsa
1924
UCGUAAGAAGUCUGAUAGAUGGA
2219

AD-292678.1
usasagaagudCudGauagauggaaL96
1630
usdTsccdAudCuaucdAgdAcuucuuascsg
1925
CGUAAGAAGUCUGAUAGAUGGAA
2220

AD-292679.1
asasgaagucdTgdAuagauggaauL96
1631
asdTsucdCadTcuaudCadGacuucuusasc
1926
GUAAGAAGUCUGAUAGAUGGAAU
2221

AD-292680.1
asgsaagucudGadTagauggaauaL96
1632
usdAsuudCcdAucuadTcdAgacuucususa
1927
UAAGAAGUCUGAUAGAUGGAAUA
2222

AD-292681.1
gsasagucugdAudAgauggaauauL96
1633
asdTsaudTcdCaucudAudCagacuucsusu
1928
AAGAAGUCUGAUAGAUGGAAUAC
2223

AD-292682.1
asasgucugadTadGauggaauacuL96
1634
asdGsuadTudCcaucdTadTcagacuuscsu
1929
AGAAGUCUGAUAGAUGGAAUACU
2224

AD-292684.1
gsuscugauadGadTggaauacuuaL96
1635
usdAsagdTadTuccadTcdTaucagacsusu
1930
AAGUCUGAUAGAUGGAAUACUUA
2225

AD-292685.1
uscsugauagdAudGgaauacuuauL96
1636
asdTsaadGudAuuccdAudCuaucagascsu
1931
AGUCUGAUAGAUGGAAUACUUAC
2226

AD-292691.1
asgsauggaadTadCuuaccaauaaL96
1637
usdTsaudTgdGuaagdTadTuccaucusasu
1932
AUAGAUGGAAUACUUACCAAUAA
2227

AD-292692.1
gsasuggaaudAcdTuaccaauaauL96
1638
asdTsuadTudGguaadGudAuuccaucsusa
1933
UAGAUGGAAUACUUACCAAUAAG
2228

AD-292693.1
asusggaauadCudTaccaauaagaL96
1639
usdCsuudAudTgguadAgdTauuccauscsu
1934
AGAUGGAAUACUUACCAAUAAGA
2229

AD-292694.1
usgsgaauacdTudAccaauaagaaL96
1640
usdTscudTadTuggudAadGuauuccasusc
1935
GAUGGAAUACUUACCAAUAAGAA
2230

AD-292695.1
gsgsaauacudTadCcaauaagaaaL96
1641
usdTsucdTudAuuggdTadAguauuccsasu
1936
AUGGAAUACUUACCAAUAAGAAA
2231

AD-292696.1
gsasauacuudAcdCaauaagaaaaL96
1642
usdTsuudCudTauugdGudAaguauucscsa
1937
UGGAAUACUUACCAAUAAGAAAA
2232

AD-292697.1
asasuacuuadCcdAauaagaaaauL96
1643
asdTsuudTcdTuauudGgdTaaguauuscsc
1938
GGAAUACUUACCAAUAAGAAAAU
2233

AD-292725.1
gsusuccaucdGudAuaucaauauuL96
1644
asdAsuadTudGauaudAcdGauggaacsasa
1939
UUGUUCCAUCGUAUAUCAAUAUC
2234

AD-292726.1
ususccaucgdTadTaucaauaucuL96
1645
asdGsaudAudTgauadTadCgauggaascsa
1940
UGUUCCAUCGUAUAUCAAUAUCU
2235

AD-292727.1
uscscaucgudAudAucaauaucuuL96
1646
asdAsgadTadTugaudAudAcgauggasasc
1941
GUUCCAUCGUAUAUCAAUAUCUU
2236

AD-292728.1
cscsaucguadTadTcaauaucuuuL96
1647
asdAsagdAudAuugadTadTacgauggsasa
1942
UUCCAUCGUAUAUCAAUAUCUUU
2237

AD-292757.1
ascsagaagudTudCuuccugaacuL96
1648
asdGsuudCadGgaagdAadAcuucugusasg
1943
CUACAGAAGUUUCUUCCUGAACG
2238

AD-292759.1
asgsaaguuudCudTccugaacgcuL96
1649
asdGscgdTudCaggadAgdAaacuucusgsu
1944
ACAGAAGUUUCUUCCUGAACGCG
2239

AD-292761.1
asasguuucudTcdCugaacgcgcuL96
1650
asdGscgdCgdTucagdGadAgaaacuuscsu
1945
AGAAGUUUCUUCCUGAACGCGCC
2240

AD-292766.1
uscsuuccugdAadCgcgccucaguL96
1651
asdCsugdAgdGcgcgdTudCaggaagasasa
1946
UUUCUUCCUGAACGCGCCUCAGC
2241

AD-292787.1
gsasuuuuaadAudCguaugcagaaL96
1652
usdTscudGcdAuacgdAudTuaaaaucsgsc
1947
GCGAUUUUAAAUCGUAUGCAGAA
2242

AD-292788.1
asusuuuaaadTcdGuaugcagaauL96
1653
asdTsucdTgdCauacdGadTuuaaaauscsg
1948
CGAUUUUAAAUCGUAUGCAGAAU
2243

AD-292789.1
ususuuaaaudCgdTaugcagaauaL96
1654
usdAsuudCudGcauadCgdAuuuaaaasusc
1949
GAUUUUAAAUCGUAUGCAGAAUA
2244

AD-292793.1
asasaucguadTgdCagaauauucaL96
1655
usdGsaadTadTucugdCadTacgauuusasa
1950
UUAAAUCGUAUGCAGAAUAUUCA
2245

AD-292795.1
asuscguaugdCadGaauauucaauL96
1656
asdTsugdAadTauucdTgdCauacgaususu
1951
AAAUCGUAUGCAGAAUAUUCAAU
2246

AD-292796.1
uscsguaugcdAgdAauauucaauuL96
1657
asdAsuudGadAuauudCudGcauacgasusu
1952
AAUCGUAUGCAGAAUAUUCAAUU
2247

AD-292797.1
csgsuaugcadGadAuauucaauuuL96
1658
asdAsaudTgdAauaudTcdTgcauacgsasu
1953
AUCGUAUGCAGAAUAUUCAAUUU
2248

AD-292800.1
asusgcagaadTadTucaauuugaaL96
1659
usdTscadAadTugaadTadTucugcausasc
1954
GUAUGCAGAAUAUUCAAUUUGAA
2249

AD-292802.1
gscsagaauadTudCaauuugaaguL96
1660
asdCsuudCadAauugdAadTauucugcsasu
1955
AUGCAGAAUAUUCAAUUUGAAGC
2250

AD-292806.1
asasuauucadAudTugaagcaguuL96
1661
asdAscudGcdTucaadAudTgaauauuscsu
1956
AGAAUAUUCAAUUUGAAGCAGUG
2251

AD-292808.1
usasuucaaudTudGaagcagugguL96
1662
asdCscadCudGcuucdAadAuugaauasusu
1957
AAUAUUCAAUUUGAAGCAGUGGU
2252

AD-292870.1
gsasuguaugdCadTgauaaugauaL96
1663
usdAsucdAudTaucadTgdCauacaucsusc
1958
GAGAUGUAUGCAUGAUAAUGAUA
2253

AD-292893.1
asasuaguuudCgdAaucaaugcuuL96
1664
asdAsgcdAudTgauudCgdAaacuauuscsa
1959
UGAAUAGUUUCGAAUCAAUGCUG
2254

AD-292906.1
csasaugcugdCadAagcuuuauuuL96
1665
asdAsaudAadAgcuudTgdCagcauugsasu
1960
AUCAAUGCUGCAAAGCUUUAUUU
2255

AD-292915.1
asasagcuuudAudTucacauuuuuL96
1666
asdAsaadAudGugaadAudAaagcuuusgsc
1961
GCAAAGCUUUAUUUCACAUUUUU
2256

AD-292935.1
asasaaacaudTgdGuuuggcacuaL96
1667
usdAsgudGcdCaaacdCadAuguuuuusasa
1962
UUAAAAACAUUGGUUUGGCACUA
2257

AD-292936.1
asasaacauudGgdTuuggcacuauL96
1668
asdTsagdTgdCcaaadCcdAauguuuususa
1963
UAAAAACAUUGGUUUGGCACUAG
2258

AD-292967.1
ascsgaacaadGadTuaauuaccuuL96
1669
asdAsggdTadAuuaadTcdTuguucgususu
1964
AAACGAACAAGAUUAAUUACCUG
2259

AD-292970.1
asascaagaudTadAuuaccugucuL96
1670
asdGsacdAgdGuaaudTadAucuuguuscsg
1965
CGAACAAGAUUAAUUACCUGUCU
2260

AD-292974.1
asgsauuaaudTadCcugucuuccuL96
1671
asdGsgadAgdAcaggdTadAuuaaucususg
1966
CAAGAUUAAUUACCUGUCUUCCU
2261

AD-292975.1
gsasuuaauudAcdCugucuuccuuL96
1672
asdAsggdAadGacagdGudAauuaaucsusu
1967
AAGAUUAAUUACCUGUCUUCCUG
2262

AD-292978.1
usasauuaccdTgdTcuuccuguuuL96
1673
asdAsacdAgdGaagadCadGguaauuasasu
1968
AUUAAUUACCUGUCUUCCUGUUU
2263

AD-292979.1
asasuuaccudGudCuuccuguuucL96
1674
gsdAsaadCadGgaagdAcdAgguaauusasa
1969
UUAAUUACCUGUCUUCCUGUUUC
2264

AD-292980.1
asusuaccugdTcdTuccuguuucuL96
1675
asdGsaadAcdAggaadGadCagguaaususa
1970
UAAUUACCUGUCUUCCUGUUUCU
2265

AD-292981.1
ususaccugudCudTccuguuucuuL96
1676
asdAsgadAadCaggadAgdAcagguaasusu
1971
AAUUACCUGUCUUCCUGUUUCUC
2266

AD-292982.1
usasccugucdTudCcuguuucucaL96
1677
usdGsagdAadAcaggdAadGacagguasasu
1972
AUUACCUGUCUUCCUGUUUCUCA
2267

AD-292983.1
ascscugucudTcdCuguuucucaaL96
1678
usdTsgadGadAacagdGadAgacaggusasa
1973
UUACCUGUCUUCCUGUUUCUCAA
2268

AD-292984.1
cscsugucuudCcdTguuucucaauL96
1679
asdTsugdAgdAaacadGgdAagacaggsusa
1974
UACCUGUCUUCCUGUUUCUCAAG
2269

AD-293027.1
gsgsucuguudTudTccuuucauguL96
1680
asdCsaudGadAaggadAadAacagaccsusa
1975
UAGGUCUGUUUUUCCUUUCAUGC
2270

AD-293028.1
gsuscuguuudTudCcuuucaugcuL96
1681
asdGscadTgdAaaggdAadAaacagacscsu
1976
AGGUCUGUUUUUCCUUUCAUGCC
2271

AD-293034.1
ususuuccuudTcdAugccucuuaaL96
1682
usdTsaadGadGgcaudGadAaggaaaasasc
1977
GUUUUUCCUUUCAUGCCUCUUAA
2272

AD-293037.1
uscscuuucadTgdCcucuuaaaaaL96
1683
usdTsuudTadAgaggdCadTgaaaggasasa
1978
UUUCCUUUCAUGCCUCUUAAAAA
2273

AD-293039.1
csusuucaugdCcdTcuuaaaaacuL96
1684
asdGsuudTudTaagadGgdCaugaaagsgsa
1979
UCCUUUCAUGCCUCUUAAAAACU
2274

AD-293099.1
ususuuuccadTudTaaagguggauL96
1685
asdTsccdAcdCuuuadAadTggaaaaasusa
1980
UAUUUUUCCAUUUAAAGGUGGAC
2275

AD-293153.1
asascuuauudTadCacagggaaguL96
1686
asdCsuudCcdCugugdTadAauaaguuscsu
1981
AGAACUUAUUUACACAGGGAAGG
2276

AD-293156.1
ususauuuacdAcdAgggaagguuuL96
1687
asdAsacdCudTcccudGudGuaaauaasgsu
1982
ACUUAUUUACACAGGGAAGGUUU
2277

AD-293162.1
ascsacagggdAadGguuuaagacuL96
1688
asdGsucdTudAaaccdTudCccugugusasa
1983
UUACACAGGGAAGGUUUAAGACU
2278

AD-293163.1
csascagggadAgdGuuuaagacuuL96
1689
asdAsgudCudTaaacdCudTcccugugsusa
1984
UACACAGGGAAGGUUUAAGACUG
2279

AD-293166.1
asgsggaaggdTudTaagacuguuuL96
1690
asdAsacdAgdTcuuadAadCcuucccusgsu
1985
ACAGGGAAGGUUUAAGACUGUUC
2280

AD-293167.1
gsgsgaaggudTudAagacuguucaL96
1691
usdGsaadCadGucuudAadAccuucccsusg
1986
CAGGGAAGGUUUAAGACUGUUCA
2281

AD-293168.1
gsgsaagguudTadAgacuguucaaL96
1692
usdTsgadAcdAgucudTadAaccuuccscsu
1987
AGGGAAGGUUUAAGACUGUUCAA
2282

AD-293169.1
gsasagguuudAadGacuguucaauL96
1693
asdTsugdAadCagucdTudAaaccuucscsc
1988
GGGAAGGUUUAAGACUGUUCAAG
2283

AD-293170.1
asasgguuuadAgdAcuguucaaguL96
1694
asdCsuudGadAcagudCudTaaaccuuscsc
1989
GGAAGGUUUAAGACUGUUCAAGU
2284

AD-293171.1
asgsguuuaadGadCuguucaaguaL96
1695
usdAscudTgdAacagdTcdTuaaaccususc
1990
GAAGGUUUAAGACUGUUCAAGUA
2285

AD-293172.1
gsgsuuuaagdAcdTguucaaguauL96
1696
asdTsacdTudGaacadGudCuuaaaccsusu
1991
AAGGUUUAAGACUGUUCAAGUAG
2286

AD-293174.1
ususuaagacdTgdTucaaguagcaL96
1697
usdGscudAcdTugaadCadGucuuaaascsc
1992
GGUUUAAGACUGUUCAAGUAGCA
2287

AD-293176.1
usasagacugdTudCaaguagcauuL96
1698
asdAsugdCudAcuugdAadCagucuuasasa
1993
UUUAAGACUGUUCAAGUAGCAUU
2288

AD-293177.1
asasgacugudTcdAaguagcauuuL96
1699
asdAsaudGcdTacuudGadAcagucuusasa
1994
UUAAGACUGUUCAAGUAGCAUUC
2289

AD-293178.1
asgsacuguudCadAguagcauucuL96
1700
asdGsaadTgdCuacudTgdAacagucususa
1995
UAAGACUGUUCAAGUAGCAUUCC
2290

AD-293179.1
gsascuguucdAadGuagcauuccaL96
1701
usdGsgadAudGcuacdTudGaacagucsusu
1996
AAGACUGUUCAAGUAGCAUUCCA
2291

AD-293180.1
ascsuguucadAgdTagcauuccaaL96
1702
usdTsggdAadTgcuadCudTgaacaguscsu
1997
AGACUGUUCAAGUAGCAUUCCAA
2292

AD-293181.1
csusguucaadGudAgcauuccaauL96
1703
asdTsugdGadAugcudAcdTugaacagsusc
1998
GACUGUUCAAGUAGCAUUCCAAU
2293

AD-293182.1
usgsuucaagdTadGcauuccaauuL96
1704
asdAsuudGgdAaugcdTadCuugaacasgsu
1999
ACUGUUCAAGUAGCAUUCCAAUC
2294

AD-293183.1
gsusucaagudAgdCauuccaaucuL96
1705
asdGsaudTgdGaaugdCudAcuugaacsasg
2000
CUGUUCAAGUAGCAUUCCAAUCU
2295

AD-293184.1
ususcaaguadGcdAuuccaaucuuL96
1706
asdAsgadTudGgaaudGcdTacuugaascsa
2001
UGUUCAAGUAGCAUUCCAAUCUG
2296

AD-293186.1
csasaguagcdAudTccaaucuguaL96
1707
usdAscadGadTuggadAudGcuacuugsasa
2002
UUCAAGUAGCAUUCCAAUCUGUA
2297

AD-293187.1
asasguagcadTudCcaaucuguauL96
1708
asdTsacdAgdAuuggdAadTgcuacuusgsa
2003
UCAAGUAGCAUUCCAAUCUGUAG
2298

AD-293191.1
asgscauuccdAadTcuguagccauL96
1709
asdTsggdCudAcagadTudGgaaugcusasc
2004
GUAGCAUUCCAAUCUGUAGCCAU
2299

AD-293218.1
gsasauaucadAcdAagaacacagaL96
1710
usdCsugdTgdTucuudGudTgauauucsusg
2005
CAGAAUAUCAACAAGAACACAGA
2300

AD-293228.1
asasgaacacdAgdAaugagugcauL96
1711
asdTsgcdAcdTcauudCudGuguucuusgsu
2006
ACAAGAACACAGAAUGAGUGCAC
2301

AD-293230.1
gsasacacagdAadTgagugcacauL96
1712
asdTsgudGcdAcucadTudCuguguucsusu
2007
AAGAACACAGAAUGAGUGCACAG
2302

AD-293231.1
asascacagadAudGagugcacaguL96
1713
asdCsugdTgdCacucdAudTcuguguuscsu
2008
AGAACACAGAAUGAGUGCACAGC
2303

AD-293235.1
csasgaaugadGudGcacagcuaauL96
1714
asdTsuadGcdTgugcdAcdTcauucugsusg
2009
CACAGAAUGAGUGCACAGCUAAG
2304

AD-293271.1
asgsgcagcudTudAucucaaccuuL96
1715
asdAsggdTudGagaudAadAgcugccusgsc
2010
GCAGGCAGCUUUAUCUCAACCUG
2305

AD-293273.1
gscsagcuuudAudCucaaccuggaL96
1716
usdCscadGgdTugagdAudAaagcugcscsu
2011
AGGCAGCUUUAUCUCAACCUGGA
2306

AD-293306.1
ususcagcaudTudGaaagauuucuL96
1717
asdGsaadAudCuuucdAadAugcugaasusc
2012
GAUUCAGCAUUUGAAAGAUUUCC
2307

AD-293309.1
asgscauuugdAadAgauuucccuaL96
1718
usdAsggdGadAaucudTudCaaaugcusgsa
2013
UCAGCAUUUGAAAGAUUUCCCUA
2308

AD-293311.1
csasuuugaadAgdAuuucccuaguL96
1719
asdCsuadGgdGaaaudCudTucaaaugscsu
2014
AGCAUUUGAAAGAUUUCCCUAGC
2309

AD-293312.1
asusuugaaadGadTuucccuagcuL96
1720
asdGscudAgdGgaaadTcdTuucaaausgsc
2015
GCAUUUGAAAGAUUUCCCUAGCC
2310

AD-293315.1
usgsaaagaudTudCccuagccucuL96
1721
asdGsagdGcdTagggdAadAucuuucasasa
2016
UUUGAAAGAUUUCCCUAGCCUCU
2311

AD-293316.1
gsasaagauudTcdCcuagccucuuL96
1722
asdAsgadGgdCuaggdGadAaucuuucsasa
2017
UUGAAAGAUUUCCCUAGCCUCUU
2312

AD-293323.1
ususcccuagdCcdTcuuccuuuuuL96
1723
asdAsaadAgdGaagadGgdCuagggaasasu
2018
AUUUCCCUAGCCUCUUCCUUUUU
2313

AD-293353.1
asasaacggudGcdAacucuauucuL96
1724
asdGsaadTadGaguudGcdAccguuuusgsg
2019
CCAAAACGGUGCAACUCUAUUCU
2314

AD-293354.1
asasacggugdCadAcucuauucuuL96
1725
asdAsgadAudAgagudTgdCaccguuususg
2020
CAAAACGGUGCAACUCUAUUCUG
2315

AD-293355.1
asascggugcdAadCucuauucuguL96
1726
asdCsagdAadTagagdTudGcaccguususu
2021
AAAACGGUGCAACUCUAUUCUGG
2316

AD-293358.1
gsgsugcaacdTcdTauucuggacuL96
1727
asdGsucdCadGaauadGadGuugcacesgsu
2022
ACGGUGCAACUCUAUUCUGGACU
2317

AD-293359.1
gsusgcaacudCudAuucuggacuuL96
1728
asdAsgudCcdAgaaudAgdAguugcacscsg
2023
CGGUGCAACUCUAUUCUGGACUU
2318

AD-293360.1
usgscaacucdTadTucuggacuuuL96
1729
asdAsagdTcdCagaadTadGaguugcascsc
2024
GGUGCAACUCUAUUCUGGACUUU
2319

AD-293361.1
gscsaacucudAudTcuggacuuuaL96
1730
usdAsaadGudCcagadAudAgaguugcsasc
2025
GUGCAACUCUAUUCUGGACUUUA
2320

AD-293362.1
csasacucuadTudCuggacuuuauL96
1731
asdTsaadAgdTccagdAadTagaguugscsa
2026
UGCAACUCUAUUCUGGACUUUAU
2321

AD-293363.1
asascucuaudTcdTggacuuuauuL96
1732
asdAsuadAadGuccadGadAuagaguusgsc
2027
GCAACUCUAUUCUGGACUUUAUU
2322

AD-293364.1
ascsucuauudCudGgacuuuauuaL96
1733
usdAsaudAadAguccdAgdAauagagususg
2028
CAACUCUAUUCUGGACUUUAUUA
2323

AD-293365.1
csuscuauucdTgdGacuuuauuauL96
1734
asdTsaadTadAagucdCadGaauagagsusu
2029
AACUCUAUUCUGGACUUUAUUAC
2324

AD-293368.1
usasuucuggdAcdTuuauuacuuuL96
1735
asdAsagdTadAuaaadGudCcagaauasgsa
2030
UCUAUUCUGGACUUUAUUACUUG
2325

AD-293369.1
asusucuggadCudTuauuacuugaL96
1736
usdCsaadGudAauaadAgdTccagaausasg
2031
CUAUUCUGGACUUUAUUACUUGA
2326

AD-293370.1
ususcuggacdTudTauuacuugauL96
1737
asdTscadAgdTaauadAadGuccagaasusa
2032
UAUUCUGGACUUUAUUACUUGAU
2327

AD-293410.1
usgsaaguccdAcdCaaaaguggauL96
1738
asdTsccdAcdTuuugdGudGgacuucasgsa
2033
UCUGAAGUCCACCAAAAGUGGAC
2328

AD-293419.1
cscsaaaagudGgdAcccucuauauL96
1739
asdTsaudAgdAgggudCcdAcuuuuggsusg
2034
CACCAAAAGUGGACCCUCUAUAU
2329

AD-293420.1
csasaaagugdGadCccucuauauuL96
1740
asdAsuadTadGagggdTcdCacuuuugsgsu
2035
ACCAAAAGUGGACCCUCUAUAUU
2330

AD-293422.1
asasaguggadCcdCucuauauuucL96
1741
gsdAsaadTadTagagdGgdTccacuuususg
2036
CAAAAGUGGACCCUCUAUAUUUC
2331

AD-293423.1
asasguggacdCcdTcuauauuucuL96
1742
asdGsaadAudAuagadGgdGuccacuususu
2037
AAAAGUGGACCCUCUAUAUUUCC
2332

AD-293530.1
csusuucauadTadTccuugguccuL96
1743
asdGsgadCcdAaggadTadTaugaaagscsa
2038
UGCUUUCAUAUAUCCUUGGUCCC
2333

AD-293532.1
ususcauauadTcdCuuggucccauL96
1744
asdTsggdGadCcaagdGadTauaugaasasg
2039
CUUUCAUAUAUCCUUGGUCCCAG
2334

AD-293554.1
gsasuguuuadGadCaauuuuagguL96
1745
asdCscudAadAauugdTcdTaaacaucsusc
2040
GAGAUGUUUAGACAAUUUUAGGC
2335

AD-293555.1
asusguuuagdAcdAauuuuaggcuL96
1746
asdGsccdTadAaauudGudCuaaacauscsu
2041
AGAUGUUUAGACAAUUUUAGGCU
2336

AD-293557.1
gsusuuagacdAadTuuuaggcucaL96
1747
usdGsagdCcdTaaaadTudGucuaaacsasu
2042
AUGUUUAGACAAUUUUAGGCUCA
2337

AD-293558.1
ususuagacadAudTuuaggcucaaL96
1748
usdTsgadGcdCuaaadAudTgucuaaascsa
2043
UGUUUAGACAAUUUUAGGCUCAA
2338

AD-293559.1
ususagacaadTudTuaggcucaaaL96
1749
usdTsugdAgdCcuaadAadTugucuaasasc
2044
GUUUAGACAAUUUUAGGCUCAAA
2339

AD-293560.1
usasgacaaudTudTaggcucaaaaL96
1750
usdTsuudGadGccuadAadAuugucuasasa
2045
UUUAGACAAUUUUAGGCUCAAAA
2340

AD-293562.1
gsascaauuudTadGgcucaaaaauL96
1751
asdTsuudTudGagccdTadAaauugucsusa
2046
UAGACAAUUUUAGGCUCAAAAAU
2341

AD-293569.1
ususaggcucdAadAaauuaaagcuL96
1752
asdGscudTudAauuudTudGagccuaasasa
2047
UUUUAGGCUCAAAAAUUAAAGCU
2342

AD-293570.1
usasggcucadAadAauuaaagcuaL96
1753
usdAsgcdTudTaauudTudTgagccuasasa
2048
UUUAGGCUCAAAAAUUAAAGCUA
2343

AD-293571.1
asgsgcucaadAadAuuaaagcuaaL96
1754
usdTsagdCudTuaaudTudTugagccusasa
2049
UUAGGCUCAAAAAUUAAAGCUAA
2344

AD-293572.1
gsgscucaaadAadTuaaagcuaauL96
1755
asdTsuadGcdTuuaadTudTuugagccsusa
2050
UAGGCUCAAAAAUUAAAGCUAAC
2345

AD-293573.1
gscsucaaaadAudTaaagcuaacaL96
1756
usdGsuudAgdCuuuadAudTuuugagcscsu
2051
AGGCUCAAAAAUUAAAGCUAACA
2346

AD-293574.1
csuscaaaaadTudAaagcuaacauL96
1757
asdTsgudTadGcuuudAadTuuuugagscsc
2052
GGCUCAAAAAUUAAAGCUAACAC
2347

AD-293575.1
uscsaaaaaudTadAagcuaacacaL96
1758
usdGsugdTudAgcuudTadAuuuuugasgsc
2053
GCUCAAAAAUUAAAGCUAACACA
2348

AD-293576.1
csasaaaauudAadAgcuaacacauL96
1759
asdTsgudGudTagcudTudAauuuuugsasg
2054
CUCAAAAAUUAAAGCUAACACAG
2349

AD-293579.1
asasauuaaadGcdTaacacaggaaL96
1760
usdTsccdTgdTguuadGcdTuuaauuususu
2055
AAAAAUUAAAGCUAACACAGGAA
2350

AD-293586.1
asgscuaacadCadGgaaaaggaauL96
1761
asdTsucdCudTuuccdTgdTguuagcususu
2056
AAAGCUAACACAGGAAAAGGAAC
2351

AD-293589.1
usasacacagdGadAaaggaacuguL96
1762
asdCsagdTudCcuuudTcdCuguguuasgsc
2057
GCUAACACAGGAAAAGGAACUGU
2352

AD-293592.1
csascaggaadAadGgaacuguacuL96
1763
asdGsuadCadGuuccdTudTuccugugsusu
2058
AACACAGGAAAAGGAACUGUACU
2353

AD-293594.1
csasggaaaadGgdAacuguacuguL96
1764
asdCsagdTadCaguudCcdTuuuccugsusg
2059
CACAGGAAAAGGAACUGUACUGG
2354

AD-293601.1
asgsgaacugdTadCuggcuauuauL96
1765
asdTsaadTadGccagdTadCaguuccususu
2060
AAAGGAACUGUACUGGCUAUUAC
2355

AD-293602.1
gsgsaacugudAcdTggcuauuacaL96
1766
usdGsuadAudAgccadGudAcaguuccsusu
2061
AAGGAACUGUACUGGCUAUUACA
2356

AD-293872.1
csgsacucccdAcdTacaucaagauL96
1767
asdTscudTgdAuguadGudGggagucgsgsa
2062
UCCGACUCCCACUACAUCAAGAC
2357

AD-293875.1
csuscccacudAcdAucaagacuaaL96
1768
usdTsagdTcdTugaudGudAgugggagsusc
2063
GACUCCCACUACAUCAAGACUAA
2358

AD-293876.1
uscsccacuadCadTcaagacuaauL96
1769
asdTsuadGudCuugadTgdTagugggasgsu
2064
ACUCCCACUACAUCAAGACUAAU
2359

AD-293877.1
cscscacuacdAudCaagacuaauuL96
1770
asdAsuudAgdTcuugdAudGuagugggsasg
2065
CUCCCACUACAUCAAGACUAAUC
2360

AD-293878.1
cscsacuacadTcdAagacuaaucuL96
1771
asdGsaudTadGucuudGadTguaguggsgsa
2066
UCCCACUACAUCAAGACUAAUCU
2361

AD-293879.1
csascuacaudCadAgacuaaucuuL96
1772
asdAsgadTudAgucudTgdAuguagugsgsg
2067
CCCACUACAUCAAGACUAAUCUU
2362

AD-293880.1
ascsuacaucdAadGacuaaucuuuL96
1773
asdAsagdAudTagucdTudGauguagusgsg
2068
CCACUACAUCAAGACUAAUCUUG
2363

AD-293881.1
csusacaucadAgdAcuaaucuuguL96
1774
asdCsaadGadTuagudCudTgauguagsusg
2069
CACUACAUCAAGACUAAUCUUGU
2364

AD-293882.1
usascaucaadGadCuaaucuuguuL96
1775
asdAscadAgdAuuagdTcdTugauguasgsu
2070
ACUACAUCAAGACUAAUCUUGUU
2365

AD-293883.1
ascsaucaagdAcdTaaucuuguuuL96
1776
asdAsacdAadGauuadGudCuugaugusasg
2071
CUACAUCAAGACUAAUCUUGUUU
2366

AD-293898.1
ususgugugudTudTucacauguauL96
1777
asdTsacdAudGugaadAadAcacacaasasa
2072
UUUUGUGUGUUUUUCACAUGUAU
2367

AD-293902.1
gsusguuuuudCadCauguauuauaL96
1778
usdAsuadAudAcaugdTgdAaaaacacsasc
2073
GUGUGUUUUUCACAUGUAUUAUA
2368

AD-293909.1
uscsacaugudAudTauagaaugcuL96
1779
asdGscadTudCuauadAudAcaugugasasa
2074
UUUCACAUGUAUUAUAGAAUGCU
2369

AD-293910.1
csascauguadTudAuagaaugcuuL96
1780
asdAsgcdAudTcuaudAadTacaugugsasa
2075
UUCACAUGUAUUAUAGAAUGCUU
2370

AD-293914.1
usgsuauuaudAgdAaugcuuuuguL96
1781
asdCsaadAadGcauudCudAuaauacasusg
2076
CAUGUAUUAUAGAAUGCUUUUGC
2371

AD-293915.1
gsusauuauadGadAugcuuuugcaL96
1782
usdGscadAadAgcaudTcdTauaauacsasu
2077
AUGUAUUAUAGAAUGCUUUUGCA
2372

AD-293919.1
usasuagaaudGcdTuuugcauggaL96
1783
usdCscadTgdCaaaadGcdAuucuauasasu
2078
AUUAUAGAAUGCUUUUGCAUGGA
2373

AD-293922.1
asgsaaugcudTudTgcauggacuaL96
1784
usdAsgudCcdAugcadAadAgcauucusasu
2079
AUAGAAUGCUUUUGCAUGGACUA
2374

AD-293923.1
gsasaugcuudTudGcauggacuauL96
1785
asdTsagdTcdCaugcdAadAagcauucsusa
2080
UAGAAUGCUUUUGCAUGGACUAU
2375

AD-293925.1
asusgcuuuudGcdAuggacuaucuL96
1786
asdGsaudAgdTccaudGcdAaaagcaususc
2081
GAAUGCUUUUGCAUGGACUAUCC
2376

AD-293928.1
csusuuugcadTgdGacuauccucuL96
1787
asdGsagdGadTagucdCadTgcaaaagscsa
2082
UGCUUUUGCAUGGACUAUCCUCU
2377

AD-293931.1
ususgcauggdAcdTauccucuuguL96
1788
asdCsaadGadGgauadGudCcaugcaasasa
2083
UUUUGCAUGGACUAUCCUCUUGU
2378

AD-293932.1
usgscauggadCudAuccucuuguuL96
1789
asdAscadAgdAggaudAgdTccaugcasasa
2084
UUUGCAUGGACUAUCCUCUUGUU
2379

AD-293933.1
gscsauggacdTadTccucuuguuuL96
1790
asdAsacdAadGaggadTadGuccaugcsasa
2085
UUGCAUGGACUAUCCUCUUGUUU
2380

AD-293934.1
csasuggacudAudCcucuuguuuuL96
1791
asdAsaadCadAgaggdAudAguccaugscsa
2086
UGCAUGGACUAUCCUCUUGUUUU
2381

AD-293935.1
asusggacuadTcdCucuuguuuuuL96
1792
asdAsaadAcdAagagdGadTaguccausgsc
2087
GCAUGGACUAUCCUCUUGUUUUU
2382

AD-293936.1
usgsgacuaudCcdTcuuguuuuuaL96
1793
usdAsaadAadCaagadGgdAuaguccasusg
2088
CAUGGACUAUCCUCUUGUUUUUA
2383

AD-293937.1
gsgsacuaucdCudCuuguuuuuauL96
1794
asdTsaadAadAcaagdAgdGauaguccsasu
2089
AUGGACUAUCCUCUUGUUUUUAU
2384

AD-293938.1
gsascuauccdTcdTuguuuuuauuL96
1795
asdAsuadAadAacaadGadGgauagucscsa
2090
UGGACUAUCCUCUUGUUUUUAUU
2385

AD-293940.1
csusauccucdTudGuuuuuauuaaL96
1796
usdTsaadTadAaaacdAadGaggauagsusc
2091
GACUAUCCUCUUGUUUUUAUUAA
2386

*Target: HSD17B13

TABLE 11

HSD17B13 NonF DNA Unmodified Sequence*

Sense
SEQ ID
Position in
Antisense
SEQ
Range in

Duplex Name
sequence 5′ to 3′
NO:
NM_178135.4
sequence 5′ to 3′
ID NO:
NM_178135.4

AD-291929.1
ACACAAGGACTGAACCAGAAU
2387
25-45
ATUCTGGUUCAGUCCUUGUGUAG
2682
23-45

AD-291931.1
ACAAGGACTGAACCAGAAGGA
2388
27-47
UCCUTCTGGUUCAGUCCUUGUGU
2683
25-47

AD-291988.1
UCCUUCUGCUTCUGAUCACCA
2389
85-105
UGGUGATCAGAAGCAGAAGGAUU
2684
83-105

AD-291993.1
CUGCUUCUGATCACCAUCAUU
2390
90-110
AAUGAUGGUGATCAGAAGCAGAA
2685
88-110

AD-291998.1
UCUGAUCACCAUCAUCUACUU
2391
95-115
AAGUAGAUGAUGGTGAUCAGAAG
2686
93-115

AD-292005.1
ACCAUCAUCUACUCCUACUUU
2392
102-122
AAAGTAGGAGUAGAUGAUGGUGA
2687
100-122

AD-292006.1
CCAUCAUCTACUCCUACUUGU
2393
103-123
ACAAGUAGGAGTAGAUGAUGGUG
2688
101-123

AD-292007.1
CAUCAUCUACTCCUACUUGGA
2394
104-124
UCCAAGTAGGAGUAGAUGAUGGU
2689
102-124

AD-292008.1
AUCAUCUACUCCUACUUGGAU
2395
105-125
ATCCAAGUAGGAGTAGAUGAUGG
2690
103-125

AD-292010.1
CAUCUACUCCTACUUGGAGUU
2396
107-127
AACUCCAAGUAGGAGUAGAUGAU
2691
105-127

AD-292012.1
UCUACUCCTACUUGGAGUCGU
2397
109-129
ACGACUCCAAGTAGGAGUAGAUG
2692
107-129

AD-292013.1
CUACUCCUACTUGGAGUCGUU
2398
110-130
AACGACTCCAAGUAGGAGUAGAU
2693
108-130

AD-292014.1
UACUCCUACUTGGAGUCGUUU
2399
111-131
AAACGACUCCAAGTAGGAGUAGA
2694
109-131

AD-292015.1
ACUCCUACTUGGAGUCGUUGU
2400
112-132
ACAACGACUCCAAGUAGGAGUAG
2695
110-132

AD-292017.1
UCCUACUUGGAGUCGUUGGUU
2402
114-134
AACCAACGACUCCAAGUAGGAGU
2697
112-134

AD-292028.1
GUCGUUGGTGAAGUUUUUCAU
2403
125-145
ATGAAAAACUUCACCAACGACUC
2698
123-145

AD-292137.1
UUGGUUCUGUGGGAUAUUAAU
2404
252-272
ATUAAUAUCCCACAGAACCAAUA
2699
250-272

AD-292140.1
GUUCUGUGGGAUAUUAAUAAU
2405
255-275
ATUATUAAUAUCCCACAGAACCA
2700
253-275

AD-292143.1
CUGUGGGATATUAAUAAGCGU
2406
258-278
ACGCTUAUUAATATCCCACAGAA
2701
256-278

AD-292182.1
GCUGAGUGCCGAAAACUAGGU
2407
297-317
ACCUAGTUUUCGGCACUCAGCUG
2702
295-317

AD-292183.1
CUGAGUGCCGAAAACUAGGCU
2408
298-318
AGCCTAGUUUUCGGCACUCAGCU
2703
296-318

AD-292185.1
GAGUGCCGAAAACUAGGCGUU
2409
300-320
AACGCCTAGUUTUCGGCACUCAG
2704
298-320

AD-292186.1
AGUGCCGAAAACUAGGCGUCA
2410
301-321
UGACGCCUAGUTUTCGGCACUCA
2705
299-321

AD-292187.1
GUGCCGAAAACUAGGCGUCAU
2411
302-322
ATGACGCCUAGTUTUCGGCACUC
2706
300-322

AD-292188.1
UGCCGAAAACTAGGCGUCACU
2412
303-323
AGUGACGCCUAGUTUUCGGCACU
2707
301-323

AD-292189.1
GCCGAAAACUAGGCGUCACUU
2413
304-324
AAGUGACGCCUAGTUUUCGGCAC
2708
302-324

AD-292190.1
CCGAAAACTAGGCGUCACUGU
2414
305-325
ACAGTGACGCCTAGUUUUCGGCA
2709
303-325

AD-292238.1
CAGAGAAGAGAUCUAUCGCUU
2415
353-373
AAGCGATAGAUCUCUUCUCUGUU
2710
351-373

AD-292244.1
AGAGAUCUAUCGCUCUCUAAA
2416
359-379
UTUAGAGAGCGAUAGAUCUCUUC
2711
357-379

AD-292245.1
GAGAUCUATCGCUCUCUAAAU
2417
360-380
ATUUAGAGAGCGATAGAUCUCUU
2712
358-380

AD-292247.1
GAUCUAUCGCTCUCUAAAUCA
2418
362-382
UGAUTUAGAGAGCGAUAGAUCUC
2713
360-382

AD-292248.1
AUCUAUCGCUCUCUAAAUCAU
2419
363-383
ATGATUTAGAGAGCGAUAGAUCU
2714
361-383

AD-292250.1
CUAUCGCUCUCUAAAUCAGGU
2420
365-385
ACCUGATUUAGAGAGCGAUAGAU
2715
363-385

AD-292251.1
UAUCGCUCTCTAAAUCAGGUU
2421
366-386
AACCTGAUUUAGAGAGCGAUAGA
2716
364-386

AD-292253.1
UCGCUCUCTAAAUCAGGUGAA
2423
368-388
UTCACCTGAUUTAGAGAGCGAUA
2718
366-388

AD-292254.1
CGCUCUCUAAAUCAGGUGAAU
2424
369-389
ATUCACCUGAUTUAGAGAGCGAU
2719
367-389

AD-292257.1
UCUCUAAATCAGGUGAAGAAA
2425
372-392
UTUCTUCACCUGATUUAGAGAGC
2720
370-392

AD-292309.1
AUGCUGGGACAGUAUAUCCAU
2426
424-444
ATGGAUAUACUGUCCCAGCAUUA
2721
422-444

AD-292310.1
UGCUGGGACAGUAUAUCCAGU
2427
425-445
ACUGGATAUACTGTCCCAGCAUU
2722
423-445

AD-292315.1
GGACAGUATATCCAGCCGAUU
2428
430-450
AAUCGGCUGGATATACUGUCCCA
2723
428-450

AD-292318.1
CAGUAUAUCCAGCCGAUCUUU
2429
433-453
AAAGAUCGGCUGGAUAUACUGUC
2724
431-453

AD-292344.1
ACCAAGGATGAAGAGAUUACU
2430
459-479
AGUAAUCUCUUCATCCUUGGUGC
2725
457-479

AD-292345.1
CCAAGGAUGAAGAGAUUACCA
2431
460-480
UGGUAATCUCUTCAUCCUUGGUG
2726
458-480

AD-292346.1
CAAGGAUGAAGAGAUUACCAA
2432
461-481
UTGGTAAUCUCTUCAUCCUUGGU
2727
459-481

AD-292363.1
CCAAGACATUTGAGGUCAACA
2433
478-498
UGUUGACCUCAAATGUCUUGGUA
2728
476-498

AD-292366.1
AGACAUUUGAGGUCAACAUCU
2434
481-501
AGAUGUTGACCTCAAAUGUCUUG
2729
479-501

AD-292367.1
GACAUUUGAGGUCAACAUCCU
2435
482-502
AGGATGTUGACCUCAAAUGUCUU
2730
480-502

AD-292369.1
CAUUUGAGGUCAACAUCCUAU
2436
484-504
ATAGGATGUUGACCUCAAAUGUC
2731
482-504

AD-292370.1
AUUUGAGGTCAACAUCCUAGU
2437
485-505
ACUAGGAUGUUGACCUCAAAUGU
2732
483-505

AD-292371.1
UUUGAGGUCAACAUCCUAGGA
2438
486-506
UCCUAGGAUGUTGACCUCAAAUG
2733
484-506

AD-292373.1
UGAGGUCAACAUCCUAGGACA
2439
488-508
UGUCCUAGGAUGUTGACCUCAAA
2734
486-508

AD-292374.1
GAGGUCAACATCCUAGGACAU
2440
489-509
ATGUCCTAGGATGTUGACCUCAA
2735
487-509

AD-292376.1
GGUCAACATCCUAGGACAUUU
2441
491-511
AAAUGUCCUAGGATGUUGACCUC
2736
489-511

AD-292377.1
GUCAACAUCCTAGGACAUUUU
2442
492-512
AAAATGTCCUAGGAUGUUGACCU
2737
490-512

AD-292379.1
CAACAUCCTAGGACAUUUUUG
2443
494-514
CAAAAATGUCCTAGGAUGUUGAC
2738
492-514

AD-292380.1
AACAUCCUAGGACAUUUUUGU
2444
495-515
ACAAAAAUGUCCUAGGAUGUUGA
2739
493-515

AD-292381.1
ACAUCCUAGGACAUUUUUGGA
2445
496-516
UCCAAAAAUGUCCTAGGAUGUUG
2740
494-516

AD-292382.1
CAUCCUAGGACAUUUUUGGAU
2446
497-517
ATCCAAAAAUGTCCUAGGAUGUU
2741
495-517

AD-292383.1
AUCCUAGGACAUUUUUGGAUU
2447
498-518
AAUCCAAAAAUGUCCUAGGAUGU
2742
496-518

AD-292384.1
UCCUAGGACATUUUUGGAUCA
2448
499-519
UGAUCCAAAAATGTCCUAGGAUG
2743
497-519

AD-292385.1
CCUAGGACAUTUUUGGAUCAU
2449
500-520
ATGATCCAAAAAUGUCCUAGGAU
2744
498-520

AD-292388.1
AGGACAUUTUTGGAUCACAAA
2450
503-523
UTUGTGAUCCAAAAAUGUCCUAG
2745
501-523

AD-292389.1
GGACAUUUTUGGAUCACAAAA
2451
504-524
UTUUGUGAUCCAAAAAUGUCCUA
2746
502-524

AD-292390.1
GACAUUUUTGGAUCACAAAAU
2452
505-525
ATUUTGTGAUCCAAAAAUGUCCU
2747
503-525

AD-292394.1
UUUUUGGATCACAAAAGCACU
2453
509-529
AGUGCUTUUGUGATCCAAAAAUG
2748
507-529

AD-292397.1
UUGGAUCACAAAAGCACUUCU
2454
512-532
AGAAGUGCUUUTGTGAUCCAAAA
2749
510-532

AD-292398.1
UGGAUCACAAAAGCACUUCUU
2455
513-533
AAGAAGTGCUUTUGUGAUCCAAA
2750
511-533

AD-292399.1
GGAUCACAAAAGCACUUCUUU
2456
514-534
AAAGAAGUGCUTUTGUGAUCCAA
2751
512-534

AD-292403.1
CACAAAAGCACUUCUUCCAUU
2457
518-538
AAUGGAAGAAGTGCUUUUGUGAU
2752
516-538

AD-292404.1
ACAAAAGCACTUCUUCCAUCU
2458
519-539
AGAUGGAAGAAGUGCUUUUGUGA
2753
517-539

AD-292405.1
CAAAAGCACUTCUUCCAUCGA
2459
520-540
UCGATGGAAGAAGTGCUUUUGUG
2754
518-540

AD-292406.1
AAAAGCACTUCUUCCAUCGAU
2460
521-541
ATCGAUGGAAGAAGUGCUUUUGU
2755
519-541

AD-292407.1
AAAGCACUTCTUCCAUCGAUU
2461
522-542
AAUCGATGGAAGAAGUGCUUUUG
2756
520-542

AD-292409.1
AGCACUUCTUCCAUCGAUGAU
2463
524-544
ATCATCGAUGGAAGAAGUGCUUU
2758
522-544

AD-292410.1
GCACUUCUTCCAUCGAUGAUU
2464
525-545
AAUCAUCGAUGGAAGAAGUGCUU
2759
523-545

AD-292434.1
AGAAAUCATGGCCACAUCGUU
2465
549-569
AACGAUGUGGCCATGAUUUCUCU
2760
547-569

AD-292486.1
UUCCUUACCUCAUCCCAUAUU
2466
601-621
AAUATGGGAUGAGGUAAGGAAUC
2761
599-621

AD-292487.1
UCCUUACCTCAUCCCAUAUUU
2467
602-622
AAAUAUGGGAUGAGGUAAGGAAU
2762
600-622

AD-292492.1
ACCUCAUCCCAUAUUGUUCCA
2468
607-627
UGGAACAAUAUGGGAUGAGGUAA
2763
605-627

AD-292495.1
UCAUCCCATATUGUUCCAGCA
2469
610-630
UGCUGGAACAATATGGGAUGAGG
2764
608-630

AD-292498.1
UCCCAUAUTGTUCCAGCAAAU
2470
613-633
ATUUGCTGGAACAAUAUGGGAUG
2765
611-633

AD-292499.1
CCCAUAUUGUTCCAGCAAAUU
2471
614-634
AAUUTGCUGGAACAAUAUGGGAU
2766
612-634

AD-292526.1
UGUUGGCUTUCACAGAGGUCU
2472
641-661
AGACCUCUGUGAAAGCCAACAGC
2767
639-661

AD-292531.1
GCUUUCACAGAGGUCUGACAU
2473
646-666
ATGUCAGACCUCUGUGAAAGCCA
2768
644-666

AD-292534.1
UUCACAGAGGTCUGACAUCAU
2474
649-669
ATGATGTCAGACCTCUGUGAAAG
2769
647-669

AD-292536.1
CACAGAGGTCTGACAUCAGAA
2475
651-671
UTCUGATGUCAGACCUCUGUGAA
2770
649-671

AD-292541.1
AGGUCUGACATCAGAACUUCA
2476
656-676
UGAAGUTCUGATGTCAGACCUCU
2771
654-676

AD-292566.1
UUGGGAAAAACUGGUAUCAAA
2477
681-701
UTUGAUACCAGTUTUUCCCAAGG
2772
679-701

AD-292574.1
AACUGGUATCAAAACCUCAUU
2478
689-709
AAUGAGGUUUUGATACCAGUUUU
2773
687-709

AD-292575.1
ACUGGUAUCAAAACCUCAUGU
2479
690-710
ACAUGAGGUUUTGAUACCAGUUU
2774
688-710

AD-292576.1
CUGGUAUCAAAACCUCAUGUU
2480
691-711
AACATGAGGUUTUGAUACCAGUU
2775
689-711

AD-292577.1
UGGUAUCAAAACCUCAUGUCU
2481
692-712
AGACAUGAGGUTUTGAUACCAGU
2776
690-712

AD-292578.1
GGUAUCAAAACCUCAUGUCUU
2482
693-713
AAGACATGAGGTUTUGAUACCAG
2777
691-713

AD-292579.1
GUAUCAAAACCUCAUGUCUCU
2483
694-714
AGAGACAUGAGGUTUUGAUACCA
2778
692-714

AD-292582.1
UCAAAACCTCAUGUCUCUGCU
2484
697-717
AGCAGAGACAUGAGGUUUUGAUA
2779
695-717

AD-292596.1
CUCUGCCCAGTUUUUGUGAAU
2485
711-731
ATUCACAAAAACUGGGCAGAGAC
2780
709-731

AD-292601.1
CCCAGUUUTUGUGAAUACUGU
2486
716-736
ACAGTATUCACAAAAACUGGGCA
2781
714-736

AD-292606.1
UUUUUGUGAATACUGGGUUCA
2487
721-741
UGAACCCAGUATUCACAAAAACU
2782
719-741

AD-292607.1
UUUUGUGAAUACUGGGUUCAU
2488
722-742
ATGAACCCAGUAUTCACAAAAAC
2783
720-742

AD-292610.1
UGUGAAUACUGGGUUCACCAA
2489
725-745
UTGGTGAACCCAGTAUUCACAAA
2784
723-745

AD-292612.1
UGAAUACUGGGUUCACCAAAA
2490
727-747
UTUUGGTGAACCCAGUAUUCACA
2785
725-747

AD-292613.1
GAAUACUGGGTUCACCAAAAA
2491
728-748
UTUUTGGUGAACCCAGUAUUCAC
2786
726-748

AD-292614.1
AAUACUGGGUTCACCAAAAAU
2492
729-749
ATUUTUGGUGAACCCAGUAUUCA
2787
727-749

AD-292615.1
AUACUGGGTUCACCAAAAAUU
2493
730-750
AAUUTUTGGUGAACCCAGUAUUC
2788
728-750

AD-292616.1
UACUGGGUTCACCAAAAAUCU
2494
731-751
AGAUTUTUGGUGAACCCAGUAUU
2789
729-751

AD-292624.1
UCACCAAAAATCCAAGCACAA
2495
739-759
UTGUGCTUGGATUTUUGGUGAAC
2790
737-759

AD-292625.1
CACCAAAAAUCCAAGCACAAU
2496
740-760
ATUGTGCUUGGAUTUUUGGUGAA
2791
738-760

AD-292629.1
AAAAAUCCAAGCACAAGAUUA
2497
744-764
UAAUCUTGUGCTUGGAUUUUUGG
2792
742-764

AD-292630.1
AAAAUCCAAGCACAAGAUUAU
2498
745-765
ATAATCTUGUGCUTGGAUUUUUG
2793
743-765

AD-292631.1
AAAUCCAAGCACAAGAUUAUU
2499
746-766
AAUAAUCUUGUGCTUGGAUUUUU
2794
744-766

AD-292634.1
UCCAAGCACAAGAUUAUGGCU
2500
749-769
AGCCAUAAUCUTGTGCUUGGAUU
2795
747-769

AD-292639.1
GCACAAGATUAUGGCCUGUAU
2502
754-774
ATACAGGCCAUAATCUUGUGCUU
2797
752-774

AD-292640.1
CACAAGAUTATGGCCUGUAUU
2503
755-775
AAUACAGGCCATAAUCUUGUGCU
2798
753-775

AD-292641.1
ACAAGAUUAUGGCCUGUAUUU
2504
756-776
AAAUACAGGCCAUAAUCUUGUGC
2799
754-776

AD-292642.1
CAAGAUUATGGCCUGUAUUGU
2505
757-777
ACAATACAGGCCATAAUCUUGUG
2800
755-777

AD-292646.1
AUUAUGGCCUGUAUUGGAGAU
2506
761-781
ATCUCCAAUACAGGCCAUAAUCU
2801
759-781

AD-292650.1
UGGCCUGUAUTGGAGACAGAU
2507
765-785
ATCUGUCUCCAAUACAGGCCAUA
2802
763-785

AD-292651.1
GGCCUGUATUGGAGACAGAUU
2508
766-786
AAUCTGTCUCCAATACAGGCCAU
2803
764-786

AD-292652.1
GCCUGUAUTGGAGACAGAUGA
2509
767-787
UCAUCUGUCUCCAAUACAGGCCA
2804
765-787

AD-292667.1
AGAUGAAGTCGUAAGAAGUCU
2510
782-802
AGACTUCUUACGACUUCAUCUGU
2805
780-802

AD-292668.1
GAUGAAGUCGTAAGAAGUCUU
2511
783-803
AAGACUTCUUACGACUUCAUCUG
2806
781-803

AD-292671.1
GAAGUCGUAAGAAGUCUGAUA
2512
786-806
UAUCAGACUUCTUACGACUUCAU
2807
784-806

AD-292673.1
AGUCGUAAGAAGUCUGAUAGA
2513
788-808
UCUATCAGACUTCTUACGACUUC
2808
786-808

AD-292677.1
GUAAGAAGTCTGAUAGAUGGA
2514
792-812
UCCATCTAUCAGACUUCUUACGA
2809
790-812

AD-292678.1
UAAGAAGUCUGAUAGAUGGAA
2515
793-813
UTCCAUCUAUCAGACUUCUUACG
2810
791-813

AD-292679.1
AAGAAGUCTGAUAGAUGGAAU
2516
794-814
ATUCCATCUAUCAGACUUCUUAC
2811
792-814

AD-292680.1
AGAAGUCUGATAGAUGGAAUA
2517
795-815
UAUUCCAUCUATCAGACUUCUUA
2812
793-815

AD-292681.1
GAAGUCUGAUAGAUGGAAUAU
2518
796-816
ATAUTCCAUCUAUCAGACUUCUU
2813
794-816

AD-292682.1
AAGUCUGATAGAUGGAAUACU
2519
797-817
AGUATUCCAUCTATCAGACUUCU
2814
795-817

AD-292684.1
GUCUGAUAGATGGAAUACUUA
2520
799-819
UAAGTATUCCATCTAUCAGACUU
2815
797-819

AD-292685.1
UCUGAUAGAUGGAAUACUUAU
2521
800-820
ATAAGUAUUCCAUCUAUCAGACU
2816
798-820

AD-292691.1
AGAUGGAATACUUACCAAUAA
2522
806-826
UTAUTGGUAAGTATUCCAUCUAU
2817
804-826

AD-292692.1
GAUGGAAUACTUACCAAUAAU
2523
807-827
ATUATUGGUAAGUAUUCCAUCUA
2818
805-827

AD-292693.1
AUGGAAUACUTACCAAUAAGA
2524
808-828
UCUUAUTGGUAAGTAUUCCAUCU
2819
806-828

AD-292694.1
UGGAAUACTUACCAAUAAGAA
2525
809-829
UTCUTATUGGUAAGUAUUCCAUC
2820
807-829

AD-292695.1
GGAAUACUTACCAAUAAGAAA
2526
810-830
UTUCTUAUUGGTAAGUAUUCCAU
2821
808-830

AD-292696.1
GAAUACUUACCAAUAAGAAAA
2527
811-831
UTUUCUTAUUGGUAAGUAUUCCA
2822
809-831

AD-292697.1
AAUACUUACCAAUAAGAAAAU
2528
812-832
ATUUTCTUAUUGGTAAGUAUUCC
2823
810-832

AD-292725.1
GUUCCAUCGUAUAUCAAUAUU
2529
840-860
AAUATUGAUAUACGAUGGAACAA
2824
838-860

AD-292726.1
UUCCAUCGTATAUCAAUAUCU
2530
841-861
AGAUAUTGAUATACGAUGGAACA
2825
839-861

AD-292727.1
UCCAUCGUAUAUCAAUAUCUU
2531
842-862
AAGATATUGAUAUACGAUGGAAC
2826
840-862

AD-292728.1
CCAUCGUATATCAAUAUCUUU
2532
843-863
AAAGAUAUUGATATACGAUGGAA
2827
841-863

AD-292757.1
ACAGAAGUTUCUUCCUGAACU
2533
872-892
AGUUCAGGAAGAAACUUCUGUAG
2828
870-892

AD-292759.1
AGAAGUUUCUTCCUGAACGCU
2534
874-894
AGCGTUCAGGAAGAAACUUCUGU
2829
872-894

AD-292761.1
AAGUUUCUTCCUGAACGCGCU
2535
876-896
AGCGCGTUCAGGAAGAAACUUCU
2830
874-896

AD-292766.1
UCUUCCUGAACGCGCCUCAGU
2536
881-901
ACUGAGGCGCGTUCAGGAAGAAA
2831
879-901

AD-292787.1
GAUUUUAAAUCGUAUGCAGAA
2537
902-922
UTCUGCAUACGAUTUAAAAUCGC
2832
900-922

AD-292788.1
AUUUUAAATCGUAUGCAGAAU
2538
903-923
ATUCTGCAUACGATUUAAAAUCG
2833
901-923

AD-292793.1
AAAUCGUATGCAGAAUAUUCA
2540
908-928
UGAATATUCUGCATACGAUUUAA
2835
906-928

AD-292795.1
AUCGUAUGCAGAAUAUUCAAU
2541
910-930
ATUGAATAUUCTGCAUACGAUUU
2836
908-930

AD-292796.1
UCGUAUGCAGAAUAUUCAAUU
2542
911-931
AAUUGAAUAUUCUGCAUACGAUU
2837
909-931

AD-292797.1
CGUAUGCAGAAUAUUCAAUUU
2543
912-932
AAAUTGAAUAUTCTGCAUACGAU
2838
910-932

AD-292800.1
AUGCAGAATATUCAAUUUGAA
2544
915-935
UTCAAATUGAATATUCUGCAUAC
2839
913-935

AD-292802.1
GCAGAAUATUCAAUUUGAAGU
2545
917-937
ACUUCAAAUUGAATAUUCUGCAU
2840
915-937

AD-292806.1
AAUAUUCAAUTUGAAGCAGUU
2546
921-941
AACUGCTUCAAAUTGAAUAUUCU
2841
919-941

AD-292870.1
GAUGUAUGCATGAUAAUGAUA
2548
989-1009
UAUCAUTAUCATGCAUACAUCUC
2843
987-1009

AD-292893.1
AAUAGUUUCGAAUCAAUGCUU
2549
1012-1032
AAGCAUTGAUUCGAAACUAUUCA
2844
1010-1032

AD-292906.1
CAAUGCUGCAAAGCUUUAUUU
2550
1025-1045
AAAUAAAGCUUTGCAGCAUUGAU
2845
1023-1045

AD-292915.1
AAAGCUUUAUTUCACAUUUUU
2551
1034-1054
AAAAAUGUGAAAUAAAGCUUUGC
2846
1032-1054

AD-292936.1
AAAACAUUGGTUUGGCACUAU
2553
1073-1093
ATAGTGCCAAACCAAUGUUUUUA
2848
1071-1093

AD-292967.1
ACGAACAAGATUAAUUACCUU
2554
1104-1124
AAGGTAAUUAATCTUGUUCGUUU
2849
1102-1124

AD-292970.1
AACAAGAUTAAUUACCUGUCU
2555
1107-1127
AGACAGGUAAUTAAUCUUGUUCG
2850
1105-1127

AD-292974.1
AGAUUAAUTACCUGUCUUCCU
2556
1111-1131
AGGAAGACAGGTAAUUAAUCUUG
2851
1109-1131

AD-292975.1
GAUUAAUUACCUGUCUUCCUU
2557
1112-1132
AAGGAAGACAGGUAAUUAAUCUU
2852
1110-1132

AD-292978.1
UAAUUACCTGTCUUCCUGUUU
2558
1115-1135
AAACAGGAAGACAGGUAAUUAAU
2853
1113-1135

AD-292979.1
AAUUACCUGUCUUCCUGUUUC
2559
1116-1136
GAAACAGGAAGACAGGUAAUUAA
2854
1114-1136

AD-292980.1
AUUACCUGTCTUCCUGUUUCU
2560
1117-1137
AGAAACAGGAAGACAGGUAAUUA
2855
1115-1137

AD-292981.1
UUACCUGUCUTCCUGUUUCUU
2561
1118-1138
AAGAAACAGGAAGACAGGUAAUU
2856
1116-1138

AD-292982.1
UACCUGUCTUCCUGUUUCUCA
2562
1119-1139
UGAGAAACAGGAAGACAGGUAAU
2857
1117-1139

AD-292983.1
ACCUGUCUTCCUGUUUCUCAA
2563
1120-1140
UTGAGAAACAGGAAGACAGGUAA
2858
1118-1140

AD-292984.1
CCUGUCUUCCTGUUUCUCAAU
2564
1121-1141
ATUGAGAAACAGGAAGACAGGUA
2859
1119-1141

AD-293027.1
GGUCUGUUTUTCCUUUCAUGU
2565
1164-1184
ACAUGAAAGGAAAAACAGACCUA
2860
1162-1184

AD-293028.1
GUCUGUUUTUCCUUUCAUGCU
2566
1165-1185
AGCATGAAAGGAAAAACAGACCU
2861
1163-1185

AD-293034.1
UUUUCCUUTCAUGCCUCUUAA
2567
1171-1191
UTAAGAGGCAUGAAAGGAAAAAC
2862
1169-1191

AD-293037.1
UCCUUUCATGCCUCUUAAAAA
2568
1174-1194
UTUUTAAGAGGCATGAAAGGAAA
2863
1172-1194

AD-293039.1
CUUUCAUGCCTCUUAAAAACU
2569
1176-1196
AGUUTUTAAGAGGCAUGAAAGGA
2864
1174-1196

AD-293099.1
UUUUUCCATUTAAAGGUGGAU
2570
1245-1265
ATCCACCUUUAAATGGAAAAAUA
2865
1243-1265

AD-293153.1
AACUUAUUTACACAGGGAAGU
2571
1299-1319
ACUUCCCUGUGTAAAUAAGUUCU
2866
1297-1319

AD-293156.1
UUAUUUACACAGGGAAGGUUU
2572
1302-1322
AAACCUTCCCUGUGUAAAUAAGU
2867
1300-1322

AD-293162.1
ACACAGGGAAGGUUUAAGACU
2573
1308-1328
AGUCTUAAACCTUCCCUGUGUAA
2868
1306-1328

AD-293163.1
CACAGGGAAGGUUUAAGACUU
2574
1309-1329
AAGUCUTAAACCUTCCCUGUGUA
2869
1307-1329

AD-293166.1
AGGGAAGGTUTAAGACUGUUU
2575
1312-1332
AAACAGTCUUAAACCUUCCCUGU
2870
1310-1332

AD-293167.1
GGGAAGGUTUAAGACUGUUCA
2576
1313-1333
UGAACAGUCUUAAACCUUCCCUG
2871
1311-1333

AD-293168.1
GGAAGGUUTAAGACUGUUCAA
2577
1314-1334
UTGAACAGUCUTAAACCUUCCCU
2872
1312-1334

AD-293169.1
GAAGGUUUAAGACUGUUCAAU
2578
1315-1335
ATUGAACAGUCTUAAACCUUCCC
2873
1313-1335

AD-293170.1
AAGGUUUAAGACUGUUCAAGU
2579
1316-1336
ACUUGAACAGUCUTAAACCUUCC
2874
1314-1336

AD-293171.1
AGGUUUAAGACUGUUCAAGUA
2580
1317-1337
UACUTGAACAGTCTUAAACCUUC
2875
1315-1337

AD-293172.1
GGUUUAAGACTGUUCAAGUAU
2581
1318-1338
ATACTUGAACAGUCUUAAACCUU
2876
1316-1338

AD-293174.1
UUUAAGACTGTUCAAGUAGCA
2582
1320-1340
UGCUACTUGAACAGUCUUAAACC
2877
1318-1340

AD-293177.1
AAGACUGUTCAAGUAGCAUUU
2584
1323-1343
AAAUGCTACUUGAACAGUCUUAA
2879
1321-1343

AD-293178.1
AGACUGUUCAAGUAGCAUUCU
2585
1324-1344
AGAATGCUACUTGAACAGUCUUA
2880
1322-1344

AD-293179.1
GACUGUUCAAGUAGCAUUCCA
2586
1325-1345
UGGAAUGCUACTUGAACAGUCUU
2881
1323-1345

AD-293180.1
ACUGUUCAAGTAGCAUUCCAA
2587
1326-1346
UTGGAATGCUACUTGAACAGUCU
2882
1324-1346

AD-293181.1
CUGUUCAAGUAGCAUUCCAAU
2588
1327-1347
ATUGGAAUGCUACTUGAACAGUC
2883
1325-1347

AD-293182.1
UGUUCAAGTAGCAUUCCAAUU
2589
1328-1348
AAUUGGAAUGCTACUUGAACAGU
2884
1326-1348

AD-293183.1
GUUCAAGUAGCAUUCCAAUCU
2590
1329-1349
AGAUTGGAAUGCUACUUGAACAG
2885
1327-1349

AD-293184.1
UUCAAGUAGCAUUCCAAUCUU
2591
1330-1350
AAGATUGGAAUGCTACUUGAACA
2886
1328-1350

AD-293186.1
CAAGUAGCAUTCCAAUCUGUA
2592
1332-1352
UACAGATUGGAAUGCUACUUGAA
2887
1330-1352

AD-293187.1
AAGUAGCATUCCAAUCUGUAU
2593
1333-1353
ATACAGAUUGGAATGCUACUUGA
2888
1331-1353

AD-293191.1
AGCAUUCCAATCUGUAGCCAU
2594
1337-1357
ATGGCUACAGATUGGAAUGCUAC
2889
1335-1357

AD-293218.1
GAAUAUCAACAAGAACACAGA
2595
1364-1384
UCUGTGTUCUUGUTGAUAUUCUG
2890
1362-1384

AD-293228.1
AAGAACACAGAAUGAGUGCAU
2596
1374-1394
ATGCACTCAUUCUGUGUUCUUGU
2891
1372-1394

AD-293230.1
GAACACAGAATGAGUGCACAU
2597
1376-1396
ATGUGCACUCATUCUGUGUUCUU
2892
1374-1396

AD-293231.1
AACACAGAAUGAGUGCACAGU
2598
1377-1397
ACUGTGCACUCAUTCUGUGUUCU
2893
1375-1397

AD-293235.1
CAGAAUGAGUGCACAGCUAAU
2599
1381-1401
ATUAGCTGUGCACTCAUUCUGUG
2894
1379-1401

AD-293271.1
AGGCAGCUTUAUCUCAACCUU
2600
1417-1437
AAGGTUGAGAUAAAGCUGCCUGC
2895
1415-1437

AD-293273.1
GCAGCUUUAUCUCAACCUGGA
2601
1419-1439
UCCAGGTUGAGAUAAAGCUGCCU
2896
1417-1439

AD-293306.1
UUCAGCAUTUGAAAGAUUUCU
2602
1452-1472
AGAAAUCUUUCAAAUGCUGAAUC
2897
1450-1472

AD-293309.1
AGCAUUUGAAAGAUUUCCCUA
2603
1455-1475
UAGGGAAAUCUTUCAAAUGCUGA
2898
1453-1475

AD-293311.1
CAUUUGAAAGAUUUCCCUAGU
2604
1457-1477
ACUAGGGAAAUCUTUCAAAUGCU
2899
1455-1477

AD-293312.1
AUUUGAAAGATUUCCCUAGCU
2605
1458-1478
AGCUAGGGAAATCTUUCAAAUGC
2900
1456-1478

AD-293315.1
UGAAAGAUTUCCCUAGCCUCU
2606
1461-1481
AGAGGCTAGGGAAAUCUUUCAAA
2901
1459-1481

AD-293323.1
UUCCCUAGCCTCUUCCUUUUU
2608
1469-1489
AAAAAGGAAGAGGCUAGGGAAAU
2903
1467-1489

AD-293353.1
AAAACGGUGCAACUCUAUUCU
2609
1499-1519
AGAATAGAGUUGCACCGUUUUGG
2904
1497-1519

AD-293354.1
AAACGGUGCAACUCUAUUCUU
2610
1500-1520
AAGAAUAGAGUTGCACCGUUUUG
2905
1498-1520

AD-293355.1
AACGGUGCAACUCUAUUCUGU
2611
1501-1521
ACAGAATAGAGTUGCACCGUUUU
2906
1499-1521

AD-293359.1
GUGCAACUCUAUUCUGGACUU
2613
1505-1525
AAGUCCAGAAUAGAGUUGCACCG
2908
1503-1525

AD-293360.1
UGCAACUCTATUCUGGACUUU
2614
1506-1526
AAAGTCCAGAATAGAGUUGCACC
2909
1504-1526

AD-293361.1
GCAACUCUAUTCUGGACUUUA
2615
1507-1527
UAAAGUCCAGAAUAGAGUUGCAC
2910
1505-1527

AD-293362.1
CAACUCUATUCUGGACUUUAU
2616
1508-1528
ATAAAGTCCAGAATAGAGUUGCA
2911
1506-1528

AD-293364.1
ACUCUAUUCUGGACUUUAUUA
2618
1510-1530
UAAUAAAGUCCAGAAUAGAGUUG
2913
1508-1530

AD-293365.1
CUCUAUUCTGGACUUUAUUAU
2619
1511-1531
ATAATAAAGUCCAGAAUAGAGUU
2914
1509-1531

AD-293368.1
UAUUCUGGACTUUAUUACUUU
2620
1514-1534
AAAGTAAUAAAGUCCAGAAUAGA
2915
1512-1534

AD-293369.1
AUUCUGGACUTUAUUACUUGA
2621
1515-1535
UCAAGUAAUAAAGTCCAGAAUAG
2916
1513-1535

AD-293370.1
UUCUGGACTUTAUUACUUGAU
2622
1516-1536
ATCAAGTAAUAAAGUCCAGAAUA
2917
1514-1536

AD-293410.1
UGAAGUCCACCAAAAGUGGAU
2623
1556-1576
ATCCACTUUUGGUGGACUUCAGA
2918
1554-1576

AD-293419.1
CCAAAAGUGGACCCUCUAUAU
2624
1565-1585
ATAUAGAGGGUCCACUUUUGGUG
2919
1563-1585

AD-293420.1
CAAAAGUGGACCCUCUAUAUU
2625
1566-1586
AAUATAGAGGGTCCACUUUUGGU
2920
1564-1586

AD-293422.1
AAAGUGGACCCUCUAUAUUUC
2626
1568-1588
GAAATATAGAGGGTCCACUUUUG
2921
1566-1588

AD-293423.1
AAGUGGACCCTCUAUAUUUCU
2627
1569-1589
AGAAAUAUAGAGGGUCCACUUUU
2922
1567-1589

AD-293530.1
CUUUCAUATATCCUUGGUCCU
2628
1699-1719
AGGACCAAGGATATAUGAAAGCA
2923
1697-1719

AD-293532.1
UUCAUAUATCCUUGGUCCCAU
2629
1701-1721
ATGGGACCAAGGATAUAUGAAAG
2924
1699-1721

AD-293554.1
GAUGUUUAGACAAUUUUAGGU
2630
1723-1743
ACCUAAAAUUGTCTAAACAUCUC
2925
1721-1743

AD-293555.1
AUGUUUAGACAAUUUUAGGCU
2631
1724-1744
AGCCTAAAAUUGUCUAAACAUCU
2926
1722-1744

AD-293557.1
GUUUAGACAATUUUAGGCUCA
2632
1726-1746
UGAGCCTAAAATUGUCUAAACAU
2927
1724-1746

AD-293558.1
UUUAGACAAUTUUAGGCUCAA
2633
1727-1747
UTGAGCCUAAAAUTGUCUAAACA
2928
1725-1747

AD-293559.1
UUAGACAATUTUAGGCUCAAA
2634
1728-1748
UTUGAGCCUAAAATUGUCUAAAC
2929
1726-1748

AD-293560.1
UAGACAAUTUTAGGCUCAAAA
2635
1729-1749
UTUUGAGCCUAAAAUUGUCUAAA
2930
1727-1749

AD-293562.1
GACAAUUUTAGGCUCAAAAAU
2636
1731-1751
ATUUTUGAGCCTAAAAUUGUCUA
2931
1729-1751

AD-293569.1
UUAGGCUCAAAAAUUAAAGCU
2637
1738-1758
AGCUTUAAUUUTUGAGCCUAAAA
2932
1736-1758

AD-293570.1
UAGGCUCAAAAAUUAAAGCUA
2638
1739-1759
UAGCTUTAAUUTUTGAGCCUAAA
2933
1737-1759

AD-293571.1
AGGCUCAAAAAUUAAAGCUAA
2639
1740-1760
UTAGCUTUAAUTUTUGAGCCUAA
2934
1738-1760

AD-293572.1
GGCUCAAAAATUAAAGCUAAU
2640
1741-1761
ATUAGCTUUAATUTUUGAGCCUA
2935
1739-1761

AD-293573.1
GCUCAAAAAUTAAAGCUAACA
2641
1742-1762
UGUUAGCUUUAAUTUUUGAGCCU
2936
1740-1762

AD-293574.1
CUCAAAAATUAAAGCUAACAU
2642
1743-1763
ATGUTAGCUUUAATUUUUGAGCC
2937
1741-1763

AD-293575.1
UCAAAAAUTAAAGCUAACACA
2643
1744-1764
UGUGTUAGCUUTAAUUUUUGAGC
2938
1742-1764

AD-293576.1
CAAAAAUUAAAGCUAACACAU
2644
1745-1765
ATGUGUTAGCUTUAAUUUUUGAG
2939
1743-1765

AD-293579.1
AAAUUAAAGCTAACACAGGAA
2645
1748-1768
UTCCTGTGUUAGCTUUAAUUUUU
2940
1746-1768

AD-293586.1
AGCUAACACAGGAAAAGGAAU
2646
1755-1775
ATUCCUTUUCCTGTGUUAGCUUU
2941
1753-1775

AD-293589.1
UAACACAGGAAAAGGAACUGU
2647
1758-1778
ACAGTUCCUUUTCCUGUGUUAGC
2942
1756-1778

AD-293592.1
CACAGGAAAAGGAACUGUACU
2648
1761-1781
AGUACAGUUCCTUTUCCUGUGUU
2943
1759-1781

AD-293594.1
CAGGAAAAGGAACUGUACUGU
2649
1763-1783
ACAGTACAGUUCCTUUUCCUGUG
2944
1761-1783

AD-293601.1
AGGAACUGTACUGGCUAUUAU
2650
1770-1790
ATAATAGCCAGTACAGUUCCUUU
2945
1768-1790

AD-293872.1
CGACUCCCACTACAUCAAGAU
2652
2186-2206
ATCUTGAUGUAGUGGGAGUCGGA
2947
2184-2206

AD-293875.1
CUCCCACUACAUCAAGACUAA
2653
2189-2209
UTAGTCTUGAUGUAGUGGGAGUC
2948
2187-2209

AD-293876.1
UCCCACUACATCAAGACUAAU
2654
2190-2210
ATUAGUCUUGATGTAGUGGGAGU
2949
2188-2210

AD-293877.1
CCCACUACAUCAAGACUAAUU
2655
2191-2211
AAUUAGTCUUGAUGUAGUGGGAG
2950
2189-2211

AD-293878.1
CCACUACATCAAGACUAAUCU
2656
2192-2212
AGAUTAGUCUUGATGUAGUGGGA
2951
2190-2212

AD-293879.1
CACUACAUCAAGACUAAUCUU
2657
2193-2213
AAGATUAGUCUTGAUGUAGUGGG
2952
2191-2213

AD-293880.1
ACUACAUCAAGACUAAUCUUU
2658
2194-2214
AAAGAUTAGUCTUGAUGUAGUGG
2953
2192-2214

AD-293881.1
CUACAUCAAGACUAAUCUUGU
2659
2195-2215
ACAAGATUAGUCUTGAUGUAGUG
2954
2193-2215

AD-293882.1
UACAUCAAGACUAAUCUUGUU
2660
2196-2216
AACAAGAUUAGTCTUGAUGUAGU
2955
2194-2216

AD-293898.1
UUGUGUGUTUTUCACAUGUAU
2662
2217-2237
ATACAUGUGAAAAACACACAAAA
2957
2215-2237

AD-293902.1
GUGUUUUUCACAUGUAUUAUA
2663
2221-2241
UAUAAUACAUGTGAAAAACACAC
2958
2219-2241

AD-293909.1
UCACAUGUAUTAUAGAAUGCU
2664
2228-2248
AGCATUCUAUAAUACAUGUGAAA
2959
2226-2248

AD-293910.1
CACAUGUATUAUAGAAUGCUU
2665
2229-2249
AAGCAUTCUAUAATACAUGUGAA
2960
2227-2249

AD-293914.1
UGUAUUAUAGAAUGCUUUUGU
2666
2233-2253
ACAAAAGCAUUCUAUAAUACAUG
2961
2231-2253

AD-293915.1
GUAUUAUAGAAUGCUUUUGCA
2667
2234-2254
UGCAAAAGCAUTCTAUAAUACAU
2962
2232-2254

AD-293919.1
UAUAGAAUGCTUUUGCAUGGA
2668
2238-2258
UCCATGCAAAAGCAUUCUAUAAU
2963
2236-2258

AD-293923.1
GAAUGCUUTUGCAUGGACUAU
2670
2242-2262
ATAGTCCAUGCAAAAGCAUUCUA
2965
2240-2262

AD-293925.1
AUGCUUUUGCAUGGACUAUCU
2671
2244-2264
AGAUAGTCCAUGCAAAAGCAUUC
2966
2242-2264

AD-293928.1
CUUUUGCATGGACUAUCCUCU
2672
2247-2267
AGAGGATAGUCCATGCAAAAGCA
2967
2245-2267

AD-293931.1
UUGCAUGGACTAUCCUCUUGU
2673
2250-2270
ACAAGAGGAUAGUCCAUGCAAAA
2968
2248-2270

AD-293932.1
UGCAUGGACUAUCCUCUUGUU
2674
2251-2271
AACAAGAGGAUAGTCCAUGCAAA
2969
2249-2271

AD-293933.1
GCAUGGACTATCCUCUUGUUU
2675
2252-2272
AAACAAGAGGATAGUCCAUGCAA
2970
2250-2272

AD-293934.1
CAUGGACUAUCCUCUUGUUUU
2676
2253-2273
AAAACAAGAGGAUAGUCCAUGCA
2971
2251-2273

AD-293935.1
AUGGACUATCCUCUUGUUUUU
2677
2254-2274
AAAAACAAGAGGATAGUCCAUGC
2972
2252-2274

AD-293936.1
UGGACUAUCCTCUUGUUUUUA
2678
2255-2275
UAAAAACAAGAGGAUAGUCCAUG
2973
2253-2275

AD-293937.1
GGACUAUCCUCUUGUUUUUAU
2679
2256-2276
ATAAAAACAAGAGGAUAGUCCAU
2974
2254-2276

AD-293938.1
GACUAUCCTCTUGUUUUUAUU
2680
2257-2277
AAUAAAAACAAGAGGAUAGUCCA
2975
2255-2277

AD-293940.1
CUAUCCUCTUGUUUUUAUUAA
2681
2259-2279
UTAATAAAAACAAGAGGAUAGUC
2976
2257-2279

*Target: HSD17B13

TABLE 12

HSD17B13 NonF DNA in vitro 10 nM screen in Cos-7 (Human Dual-Luciferase

psiCHECK2 vector) and Primary Cynomolgus Hepatocytes

% of Human Message
% of Human Message
% of Cyno Message
% of Cyno Message

Duplex Name
Remaining 10 nM
Remaining 0.1 nM
remaining 10 nM
remaining 0.1 nM

AD-291929.1
71.9
80.9
83.6
83.4

AD-291931.1
110.8
84.8
100.1
97.8

AD-291988.1
78.0
104.0
65.3
97.3

AD-291993.1
102.1
100.5
78.9
69.1

AD-291998.1
81.6
97.4
26.3
60.9

AD-292005.1
101.3
93.7
69.2
74.9

AD-292006.1
45.7
93.2
58.7
81.7

AD-292007.1
130.7
101.5
62.4
100.0

AD-292008.1
99.4
102.4
63.7
91.3

AD-292010.1
72.6
87.1
49.5
95.8

AD-292012.1
79.5
83.6
86.8
86.3

AD-292013.1
61.3
94.1
45.2
83.9

AD-292014.1
50.0
87.4
40.0
40.1

AD-292015.1
44.9
88.2
31.6
78.8

AD-292016.1
52.6
84.2
48.2
90.8

AD-292017.1
43.8
86.8
54.8
96.7

AD-292028.1
77.4
88.2
107.2
91.5

AD-292137.1
96.9
83.6
105.7
103.9

AD-292140.1
30.6
95.0
26.3
59.0

AD-292143.1
38.4
79.3
33.9
85.3

AD-292182.1
127.2
99.8
91.8
71.1

AD-292183.1
95.2
91.8
102.5
87.6

AD-292185.1
50.6
91.7
74.5
93.3

AD-292186.1
48.6
95.0
81.3
85.6

AD-292187.1
90.1
89.5
82.7
95.5

AD-292188.1
53.1
99.0
42.5
81.8

AD-292189.1
103.9
78.7
73.2
104.0

AD-292190.1
67.5
81.6
105.5
81.9

AD-292238.1
40.6
86.1
71.7
86.6

AD-292244.1
92.8
78.7
116.0
91.8

AD-292245.1
62.2
75.2
55.5
97.0

AD-292247.1
51.0
84.4
63.4
87.5

AD-292248.1
77.4
104.3
51.0
94.8

AD-292250.1
31.0
85.4
44.9
92.2

AD-292251.1
45.7
91.0
74.1
90.6

AD-292252.1
60.0
83.4
88.6
102.1

AD-292253.1
29.1
121.4
35.3
75.7

AD-292254.1
50.1
94.7
32.3
62.3

AD-292257.1
54.9
96.3
57.6
84.8

AD-292309.1
93.2
108.1
93.4
87.1

AD-292310.1
94.4
101.0
87.9
101.6

AD-292315.1
86.1
88.8
62.9
90.8

AD-292318.1
67.1
105.0
85.3
85.5

AD-292344.1
36.1
91.7
33.0
53.0

AD-292345.1
68.5
88.1
81.9
106.0

AD-292346.1
38.8
80.4
63.7
66.2

AD-292363.1
85.2
76.8
96.0
101.8

AD-292366.1
75.3
82.9
100.2
107.5

AD-292367.1
86.9
70.2
97.0
113.8

AD-292369.1
96.6
88.6
91.3
91.6

AD-292370.1
44.8
84.8
59.7
88.9

AD-292371.1
47.1
76.8
78.3
96.3

AD-292373.1
83.4
79.9
32.3
128.5

AD-292374.1
126.7
92.9
78.8
98.5

AD-292376.1
63.8
89.3
73.9
87.3

AD-292377.1
47.5
79.9
49.7
77.6

AD-292379.1
92.9
99.8
99.0
86.5

AD-292380.1
69.1
82.6
46.5
70.2

AD-292381.1
30.1
73.4
58.1
95.4

AD-292382.1
42.1
86.7
87.3
97.4

AD-292383.1
18.7
69.7
47.1
41.7

AD-292384.1
27.4
83.2
36.6
83.8

AD-292385.1
59.5
107.0
51.1
115.1

AD-292388.1
33.9
80.2
37.4
51.4

AD-292389.1
54.9
90.8
79.0
92.4

AD-292390.1
43.4
81.4
49.8
82.4

AD-292394.1
20.8
74.8
56.3
57.0

AD-292397.1
87.6
80.3
56.8
79.6

AD-292398.1
25.3
84.3
21.9
56.5

AD-292399.1
43.1
84.4
26.6
61.1

AD-292403.1
75.2
91.7
19.6
59.7

AD-292404.1
99.9
89.7
68.5
70.8

AD-292405.1
65.8
93.2
39.0
49.5

AD-292406.1
77.3
70.4
89.7
96.8

AD-292407.1
47.4
91.5
64.1
50.3

AD-292408.1
67.3
102.5
72.1
89.8

AD-292409.1
47.3
84.2
88.6
91.7

AD-292410.1
90.3
89.2
50.5
94.3

AD-292434.1
109.2
85.6
97.7
118.1

AD-292486.1
87.6
93.4
81.4
70.9

AD-292487.1
71.1
91.4
83.5
101.3

AD-292492.1
55.7
89.0
83.1
104.7

AD-292495.1
74.2
87.6
39.8
89.4

AD-292498.1
59.5
94.1
48.3
102.0

AD-292499.1
40.6
95.4
36.7
69.9

AD-292526.1
82.6
90.8
66.4
71.7

AD-292531.1
95.5
81.2
100.4
110.5

AD-292534.1
78.2
88.3
60.2
76.1

AD-292536.1
55.9
90.9
43.0
82.7

AD-292541.1
88.3
74.4
27.8
41.9

AD-292566.1
19.9
72.5
13.2
52.6

AD-292574.1
24.4
71.6
26.1
73.5

AD-292575.1
65.6
81.9
77.2
81.2

AD-292576.1
16.5
80.7
28.5
43.3

AD-292577.1
43.3
95.8
25.8
68.5

AD-292578.1
23.4
58.8
15.6
68.8

AD-292579.1
39.4
80.5
18.5
81.3

AD-292582.1
53.7
89.0
56.3
96.1

AD-292596.1
71.7
90.7
34.6
78.4

AD-292601.1
80.9
103.8
83.1
101.7

AD-292606.1
72.8
93.1
71.1
110.5

AD-292607.1
64.0
94.9
96.3
81.4

AD-292610.1
76.6
83.1
88.3
90.3

AD-292612.1
52.9
81.4
66.2
81.5

AD-292613.1
27.9
79.6
53.3
96.2

AD-292614.1
51.4
92.8
87.1
96.4

AD-292615.1
75.1
88.2
92.9
108.1

AD-292616.1
73.9
90.6
90.3
85.7

AD-292624.1
54.2
97.1
59.3
99.4

AD-292625.1
28.1
83.1
22.7
73.4

AD-292629.1
25.7
80.1
49.1
63.6

AD-292630.1
26.4
99.1
31.1
47.4

AD-292631.1
53.9
70.9
84.9
103.8

AD-292634.1
74.5
92.9
89.3
82.6

AD-292638.1
50.1
88.5
74.8
79.7

AD-292639.1
73.9
86.3
62.4
85.5

AD-292640.1
17.0
71.2
49.7
48.3

AD-292641.1
55.1
100.4
70.5
92.6

AD-292642.1
29.7
93.0
50.8
81.0

AD-292646.1
41.7
95.5
79.8
92.7

AD-292650.1
85.5
84.2
104.5
95.4

AD-292651.1
44.4
94.8
72.9
84.0

AD-292652.1
78.7
85.1
73.9
97.5

AD-292667.1
91.8
83.0
108.4
95.8

AD-292668.1
24.5
61.0
95.9
83.9

AD-292671.1
18.8
61.8
42.5
93.0

AD-292673.1
41.0
77.7
35.8
79.4

AD-292677.1
73.0
74.0
111.0
90.7

AD-292678.1
60.3
90.4
56.9
95.8

AD-292679.1
45.0
74.8
45.1
72.5

AD-292680.1
42.9
89.4
22.9
44.0

AD-292681.1
56.4
99.9
94.6
105.1

AD-292682.1
63.9
84.7
64.0
64.5

AD-292684.1
24.2
81.8
16.2
88.1

AD-292685.1
55.6
89.3
40.6
81.3

AD-292691.1
27.0
82.0
27.8
59.7

AD-292692.1
56.9
76.9
57.8
85.9

AD-292693.1
24.3
76.9
16.6
47.8

AD-292694.1
26.5
84.2
61.0
91.0

AD-292695.1
35.8
82.1
24.0
62.8

AD-292696.1
32.5
87.4
27.5
60.8

AD-292697.1
51.8
79.1
33.8
57.3

AD-292725.1
49.1
61.8
101.5
90.3

AD-292726.1
41.3
71.5
81.7
90.6

AD-292727.1
64.4
82.9
78.5
96.0

AD-292728.1
54.9
65.4
96.9
121.5

AD-292757.1
38.0
71.0
53.7
72.0

AD-292759.1
93.8
89.4
89.7
82.5

AD-292761.1
47.1
97.8
29.0
84.6

AD-292766.1
139.5
89.6
92.1
105.5

AD-292787.1
57.9
77.4
79.6
95.3

AD-292788.1
23.2
88.1
75.5
85.9

AD-292789.1
14.8
78.8
47.7
73.0

AD-292793.1
16.8
62.8
16.1
33.1

AD-292795.1
14.3
57.5
53.5
43.0

AD-292796.1
22.7
56.2
18.2
71.8

AD-292797.1
5.6
28.2
10.4
17.0

AD-292800.1
9.6
58.3
12.8
25.3

AD-292802.1
13.0
70.4
18.8
60.5

AD-292806.1
14.9
63.8
9.9
39.7

AD-292808.1
76.4
90.2
64.5
85.2

AD-292870.1
11.8
42.5
33.1
50.4

AD-292893.1
19.5
60.5
126.3
95.7

AD-292906.1
26.3
32.8
39.7
80.9

AD-292915.1
19.4
49.0
41.4
38.1

AD-292935.1
73.8
82.0
42.3
64.3

AD-292936.1
60.6
74.3
51.1
53.1

AD-292967.1
11.4
46.8
51.2
89.8

AD-292970.1
17.0
58.6
48.2
87.4

AD-292974.1
23.3
72.1
68.3
78.4

AD-292975.1
22.3
80.7
52.6
79.2

AD-292978.1
23.6
77.6
29.9
74.0

AD-292979.1
50.5
84.9
54.2
97.1

AD-292980.1
36.4
107.7
42.5
91.0

AD-292981.1
67.7
79.8
76.1
107.9

AD-292982.1
27.1
76.5
42.7
74.1

AD-292983.1
21.5
71.3
39.1
77.3

AD-292984.1
24.2
73.5
36.2
69.6

AD-293027.1
37.3
82.5
41.7
86.2

AD-293028.1
59.8
81.3
64.4
92.3

AD-293034.1
30.5
89.4
40.2
54.5

AD-293037.1
45.8
77.7
42.3
81.9

AD-293039.1
25.3
76.7
37.8
49.6

AD-293099.1
47.1
87.8
50.8
77.5

AD-293153.1
71.0
85.3
75.2
104.1

AD-293156.1
38.3
93.0
46.6
131.7

AD-293162.1
53.5
82.3
65.5
99.4

AD-293163.1
36.1
84.2
74.8
96.1

AD-293166.1
55.4
86.7
56.3
79.2

AD-293167.1
69.9
94.9
52.4
63.0

AD-293168.1
68.5
77.5
62.5
74.1

AD-293169.1
62.0
89.4
51.6
98.0

AD-293170.1
27.7
93.7
39.6
64.8

AD-293171.1
14.1
67.7
42.0
36.4

AD-293172.1
63.2
82.8
64.9
88.2

AD-293174.1
19.3
95.6
50.2
68.6

AD-293176.1
20.5
63.7
36.4
40.1

AD-293177.1
21.4
93.0
31.5
44.8

AD-293178.1
24.3
73.6
70.8
75.4

AD-293179.1
52.5
78.7
51.1
85.3

AD-293180.1
34.0
91.7
60.7
71.5

AD-293181.1
58.7
107.6
35.6
78.0

AD-293182.1
16.9
60.5
39.5
45.0

AD-293183.1
31.4
78.1
74.7
83.4

AD-293184.1
25.6
58.7
59.7
50.9

AD-293186.1
48.4
78.2
41.4
73.5

AD-293187.1
41.7
78.8
50.4
84.9

AD-293191.1
56.1
103.5
72.3
84.1

AD-293218.1
36.6
67.3
44.9
69.3

AD-293228.1
83.7
92.4
84.0
74.2

AD-293230.1
76.4
95.3
76.2
67.1

AD-293231.1
57.6
83.2
56.3
101.8

AD-293235.1
56.7
97.6
99.6
86.3

AD-293271.1
112.7
85.7
85.7
110.6

AD-293273.1
60.2
92.6
38.6
89.9

AD-293306.1
33.9
90.2
42.6
70.4

AD-293309.1
28.6
68.9
33.4
58.9

AD-293311.1
13.4
66.4
28.2
41.2

AD-293312.1
21.7
88.7
46.9
92.5

AD-293315.1
29.4
88.6
50.1
82.0

AD-293316.1
32.7
75.3
40.2
67.0

AD-293323.1
85.3
103.1
49.1
91.3

AD-293353.1
64.0
76.6
83.0
84.7

AD-293354.1
50.9
79.2
64.5
84.5

AD-293355.1
78.0
110.8
76.0
88.7

AD-293358.1
46.0
106.3
91.5
98.3

AD-293359.1
77.0
73.5
74.2
82.4

AD-293360.1
75.9
104.8
56.5
88.2

AD-293361.1
73.4
87.3
72.7
86.0

AD-293362.1
38.3
81.1
75.0
84.3

AD-293363.1
35.2
72.6
78.9
79.4

AD-293364.1
83.5
86.1
80.9
74.2

AD-293365.1
55.0
88.5
69.3
114.8

AD-293368.1
19.6
50.5
40.7
66.8

AD-293369.1
14.3
63.5
38.5
63.5

AD-293370.1
93.8
84.2
99.4
112.0

AD-293410.1
99.6
81.5
111.6
112.2

AD-293419.1
84.8
89.5
88.7
66.7

AD-293420.1
65.4
89.3
72.7
75.8

AD-293422.1
55.1
104.1
99.0
97.0

AD-293423.1
58.8
75.0
82.0
101.6

AD-293530.1
33.5
86.1
54.2
103.1

AD-293532.1
42.7
95.8
84.4
91.4

AD-293554.1
63.0
84.0
106.1
100.8

AD-293555.1
49.0
87.7
85.4
110.5

AD-293557.1
34.8
90.9
42.7
71.2

AD-293558.1
34.0
63.5
56.9
62.9

AD-293559.1
23.4
53.8
52.4
48.2

AD-293560.1
23.8
75.6
45.8
46.4

AD-293562.1
54.3
67.3
45.0
68.8

AD-293569.1
54.2
99.4
144.4
123.6

AD-293570.1
43.8
106.2
66.9
67.3

AD-293571.1
17.8
67.2
45.3
58.4

AD-293572.1
25.4
78.2
43.2
52.5

AD-293573.1
33.1
88.7
43.2
61.4

AD-293574.1
89.3
84.3
70.6
94.1

AD-293575.1
50.3
80.2
50.3
73.4

AD-293576.1
47.3
88.6
42.2
69.7

AD-293579.1
37.0
88.6
43.0
78.7

AD-293586.1
109.6
101.6
69.8
111.1

AD-293589.1
66.1
82.7
86.4
104.3

AD-293592.1
54.7
84.6
63.6
95.7

AD-293594.1
41.8
82.8
53.7
80.9

AD-293601.1
40.1
80.8
69.9
68.9

AD-293602.1
78.8
77.2
75.2
72.3

AD-293872.1
89.1
88.4
106.8
116.8

AD-293875.1
74.4
78.5
78.7
84.3

AD-293876.1
25.1
68.3
70.4
61.4

AD-293877.1
26.8
77.7
35.9
50.4

AD-293878.1
13.3
80.2
75.0
64.2

AD-293879.1
34.1
59.4
45.2
54.3

AD-293880.1
19.0
80.8
59.2
55.4

AD-293881.1
16.3
78.2
38.7
49.0

AD-293882.1
11.9
39.5
33.7
47.3

AD-293883.1
8.9
58.7
50.9
34.1

AD-293898.1
23.0
64.6
41.5
76.4

AD-293902.1
16.0
53.9
43.3
56.9

AD-293909.1
75.9
112.6
74.2
91.0

AD-293910.1
32.6
89.6
54.3
79.9

AD-293914.1
35.0
88.0
52.9
72.0

AD-293915.1
22.7
87.0
39.4
82.5

AD-293919.1
31.8
79.6
59.5
84.2

AD-293922.1
53.2
96.2
37.0
65.9

AD-293923.1
48.5
76.0
77.7
99.7

AD-293925.1
31.1
81.9
53.0
87.7

AD-293928.1
29.4
73.5
68.8
95.5

AD-293931.1
27.5
85.4
52.5
79.1

AD-293932.1
14.2
61.7
55.5
65.7

AD-293933.1
49.1
80.3
42.1
74.9

AD-293934.1
30.9
79.7
46.1
87.7

AD-293935.1
48.7
79.1
68.3
70.8

AD-293936.1
55.0
84.6
45.8
90.3

AD-293937.1
29.6
80.1
58.5
83.9

AD-293938.1
24.7
67.9
58.4
94.8

AD-293940.1
17.7
61.9
51.9
69.0

Example 3: Identification of a Loss-Of-Function Mutation that Protects Agains NASH

Non-alcoholic steatohepatitis (NASH) is a progressive disease characterized by hepatic fat build-up and inflammation that may lead to cirrhosis. NASH is predicted to be the major driver for liver transplantation by 2020. Liver-related mortality increases exponentially with fibrosis progression.

A splice variant (rs72613567:TA) in HSD17B13 results in an unstable and truncated protein with reduced enzymatic activity against steroid substrates and, thus, represents a loss-of function variant in HSD17B13. HSD17B13:rs72613567:TA was reproducibly associated with reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and as depicted in FIG. 1, this variant is associated with significantly reduced risk of alcoholic and nonalcoholic liver disease, cirrhosis and hepatocellular carcinoma in an allele dosage-dependent manner.

HSD17B13, expressed in hepatocytes, is, thus, an attractive target for the treatment of NASH and other chronic liver diseases using dsRNA agents that inhibit the expression of HSD17B13 and mimic the loss-of-function variant rs72613567:TA.

Example 4: In Vivo Efficacy of dsRNA Agents Targeting HSD17B13

A. Human/NHP Cross-Reactive siRNA Efficacy Against Ectopically Expressed Human HSD17B13 in Mice

Mice (n=3/group) were injected with an adeno-associated virus (AAV) expressing human HSD17B13. Fourteen days after AAV injection, mice were subcutaneously administered a single 3 mg/kg dose of AD-288917. Ten days after administration, human HSD17B13 expression in liver was determined by RT-qPCR and normalized against human HSD17B13 expression of control animals injected with PBS. As depicted in FIG. 2A, a single dose of AD-288917 effectively inhibits the expression of human HSD17B13.

B. Human/NHP Cross-Reactive siRNA Efficacy Against Endogenous HSD17B13 in Non-Human Primates

Cynomolgus monkeys (n=3/group) were subcutaneously administered a single 3 mg/kg dose of AD-288917. Twenty-one days after duplex 1 administration, endogenous HSD17B13 expression was determined in liver biopsies by RT-qPCR and normalized against endogenous HSD17B13 expression of control animals injected with PBS. As depicted in FIG. 2B, a single dose of AD-288917 effectively inhibits the expression of endogenous HSD17B13.

Example 5: In Vivo Efficacy of dsRNA Agents Targeting HSD17B13

Cynomolgus monkeys (n=3/group) were subcutaneously administered a single 3 mg/kg dose or a single 10 mg/kg dose of an agent listed in Table 13. Twenty-one days after duplex administration, endogenous HSD17B13 expression was determined in liver biopsies (left and right lobes) by RT-qPCR and normalized against endogenous HSD17B13 expression of control animals injected with PBS. As shown in Table 14, a single 3 mg/kg or 10 mg/kg dose of all of the agents tested effectively inhibited the expression of endogenous HSD17B 13.

TABLE 14

3 mg/kg
10 mg/kg

Groups
Left
Right
Average
Left
Right
Average

(n = 3/group)
lobe
lobe
L + R
lobe
lobe
L + R

AD-288917 AVG
0.58
0.58
0.58
0.25
0.26
0.25

AD-288917 SEM
0.26
0.26
0.26
0.01
0.05
0.03

AD-288996 AVG
0.28
0.18
0.23
0.11
0.11
0.11

AD-288996 SEM
0.08
0.09
0.07
0.06
0.05
0.05

AD-413639 AVG
0.35
0.34
0.34
0.14
0.13
0.13

AD-413639 SEM
0.16
0.11
0.13
0.07
0.06
0.06

AD-413644 AVG
0.33
0.30
0.32
0.10
0.11
0.11

AD-413644 SEM
0.13
0.09
0.10
0.02
0.03
0.02

AD-413669 AVG
0.33
0.23
0.28
0.23
0.20
0.21

AD-413669 SEM
0.09
0.07
0.08
0.11
0.10
0.10

PBS (control) AVG
1.06
1.02
1.04

PBS (control) SEM
0.26
0.20
0.23

Results represent HSD17B13 expression (fold change versus PBS-control group)

TABLE 13

SEQ

SEQ

ID
Unmodified
ID

Duplex ID
Strand
Modified Sequence (5′-3′)
NO:
Sequenceq (5′-3′)
NO:

AD-288917
sense
usascaucAfaGfAfCfuaaucuuguuL96
4486
UACAUCAAGACUAAUCUUGUU
4496

antisense
asAfscaag(Agn)uuagucUfuGfauguasgsu
4487
AACAAGAUUAGUCUUGAUGUAGU
4497

AD-288996
sense
asusgcuuUfuGfCfAfuggacuaucuL96
4488
AUGCUUUUGCAUGGACUAUCU
4498

antisense
asGfsauag(Tgn)ccaugcAfaAfagcaususc
4489
AGAUAGTCCAUGCAAAAGCAUUC
4499

AD-413639
sense
csgsuaugCfaGfAfAfuauucaauuuL96
4490
CGUAUGCAGAAUAUUCAAUUU
4500

antisense
asAfsauuGf(Agn)auauucUfgCfauacgsasu
4491
AAAUUGAAUAUUCUGCAUACGAU
4501

AD-413644
sense
csgsuaugCfaGfAfAfuauucaauuuL96
4492
CGUAUGCAGAAUAUUCAAUUU
4502

antisense
asAfsaUfuGf(Agn)auauUfcUfgCfaUfaCfgsasu
4493
AAAUUGAAUAUUCUGCAUACGAU
4503

AD-413669
sense
csusacauCfaAfGfAfcuaaucuuguL96
4494
CUACAUCAAGACUAAUCUUGU
4504

antisense
asCfsaaga(Tgn)uaguCfuUfgAfuguagsusg
4495
ACAAGATUAGUCUUGAUGUAGUG
4505

Number	Date	Country
62645941	Mar 2018	US
62770298	Nov 2018	US
62775590	Dec 2018	US

17ß-Hydroxysteroid Dehydrogenase Type 13 (HSD17B13) iRNA Compositions And Methods Of Use Thereof

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